Inhibitors of long and very long chain fatty acid metabolism as broad spectrum anti-virals

ABSTRACT

The present invention provides methods and compounds for treating viral infections using modulators of host cell enzymes relating to long chain fatty acid and lipid droplet metabolism. It includes a method of treating viral infections using triacsin C and its relatives, analogues and derivatives as well as other inhibitors of long chain fatty acid metabolism and lipid droplet metabolism.

STATEMENT OF GOVERNMENT INTEREST

The invention was made with United States Government support under GrantNos. AI068678 and CA85786 awarded by the National Institutes of Health.The Government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention provides methods and compounds for treating viralinfections using modulators of host cell enzymes relating to long chainfatty acid and lipid droplet metabolism. It includes a method oftreating viral infections using triacsin C and its relatives, analoguesand derivatives as well as other inhibitors of long chain fatty acidmetabolism and lipid droplet metabolism.

BACKGROUND OF THE INVENTION

There is a great unmet medical need for agents that more safely,effectively, and reliably treat viral infections, from HIV to the commoncold. This includes a major need for better agents to treat humancytomegalovirus (where current agents suffer from significant toxicityand lack of efficacy), herpes simplex virus (where current agents arebeneficial but provide incomplete relief), influenza A (where resistanceto current agents is rampant), and hepatitis C virus (where manypatients die from poor disease control). It further includes a majorneed for agents that work across a spectrum of viruses, facilitatingtheir clinical use without necessarily requiring identification of theunderlying pathogen.

SUMMARY OF THE INVENTION

The invention provides a method of treating or preventing viralinfection in a mammal, comprising administering to a mammalian subjectin need thereof a therapeutically effective amount of a compound orprodrug thereof, or pharmaceutically acceptable salt or ester of saidcompound or prodrug, wherein the compound is a compound of Formula I:

wherein R¹ is a carbon chain having from 3 to 23 atoms (includingoptional heteroatoms) in the chain, wherein the chain comprises 0-10double bonds within the chain, and 0-4 heteroatoms within the chain, andwherein 0-8 of the carbon atoms of R¹ are optionally substituted. If oneor more optional heteroatoms occur within the R¹ chain, in preferredembodiments each heteroatom is independently selected from O, S, andNR², wherein R² is selected from H, C₁₋₆ alkyl, and C₃₋₆ cycloalkyl.When the carbon atoms of R¹ are substituted, it is preferred that from0-8 hydrogen atoms along the chain may be replaced by a substituentselected from halo, OR², SR², lower alkyl, and cycloalkyl, wherein R² isH, C₁₋₆ alkyl, and C₃₋₆ cycloalkyl. In certain preferred embodiments, R¹is unsubstituted (i.e., R¹ is unbranched, and none of the hydrogens havebeen replaced by a substituent). In preferred embodiments for compoundsof the formula I, R¹ has a chain length of 8 to 12 atoms. Morepreferably, R¹ has a total chain length of R¹ has a chain length of 9 to11 atoms. Most preferably R¹ has a chain length of 10 atoms. In otherpreferred embodiments, R¹ has 2 to 4 double bonds. In another embodimentof the invention, the compound of Formula I is a triacsin. In yetanother embodiment of the invention, the compound of Formula I istriacsin C. In still another embodiment of the invention, the compoundis an inhibitor of acyl-CoA synthetase long-chain family member 1(ACSL1). In still another embodiment of the invention, the compound isan inhibitor of another enzyme inhibited by triacsin C, includingwithout limitation arachidonoyl-CoA synthase or enzymes of triglyceride(TG) or cholesterol ester (CE) synthesis.

Compounds of Formula I are broad spectrum antivirals useful for treatingor preventing infection by a wide range of viruses. In one embodiment,the compounds of Formula I are used to treat or prevent viral infectionby an enveloped virus. In certain embodiments of the invention, thecompounds are used to treat or prevent infection by humancytomegalovirus (HCMV), herpes simplex virus-1 (HSV-1), influenza A, orhepatitis C virus.

The invention also provides a pharmaceutical composition for treatmentor prevention of a viral infection comprising a therapeuticallyeffective amount of a composition comprising a compound or prodrugthereof, or pharmaceutically acceptable salt of said compound orprodrug; and a pharmaceutically acceptable carrier, wherein the compoundis a compound of Formula I.

In one embodiment, the invention provides a method of treating orpreventing viral infection in a mammal, comprising administering to amammalian subject in need thereof a therapeutically effective amount ofa compound or prodrug thereof, or pharmaceutically acceptable salt orester of said compound or prodrug, wherein the compound is a compound offormula V

wherein

-   X and Y are independently selected from N and CH;-   R_(1′) nd R_(2′) are independently selected from H, C₁₋₆ alkyl which    may be optionally substituted with F, OCH₃ and OH, and C₁₋₆    cycloalkyl;-   R₆ and R₇ are independently selected from H, and C₁₋₃ alkyl, or R₆    and R₇ taken together may form a C3-6 cycloalkyl,-   R₃, R₄ and R₅ are independently selected from H, C₁₋₆ alkyl which    may be optionally substituted with F, OCH₃ and OH, and C₁₋₆    cycloalkyl;-   additionally or alternatively, one of R₆ or R₇ may be taken together    with R₅ to form a C₅₋₁₁ cycloalkyl ring.

In one embodiment, the compound of formula V is avasimibe. In oneembodiment the compound of formula V is an inhibitor of Acyl-CoAcholesterol acyltransferase (ACAT).

In one embodiment, the invention provides a method of treating orpreventing viral infection in a mammal, comprising administering to amammalian subject in need thereof a therapeutically effective amount ofa compound or prodrug thereof, or pharmaceutically acceptable salt orester of said compound or prodrug, wherein the compound is selected fromthe group consisting of eflucimibe, pactimibe, Compound 1, Compound 21,Compound 12g, SMP-797, CL-283,546, and Wu-V-23. In one embodiment thecompound inhibitor of Acyl-CoA cholesterol acyltransferase (ACAT).

In one embodiment, the invention provides a method of treating orpreventing viral infection in a mammal, comprising administering to amammalian subject in need thereof a therapeutically effective amount ofa compound or prodrug thereof, or pharmaceutically acceptable salt orester of said compound or prodrug, wherein the compound is selected fromthe group consisting of PF-1052, spylidone, rubimaillin, sespendole,terpendole C, Compound 7, Compound 8, Compound 9, vermisporin,beauveriolides, phenochalasins, isobisvertinol, and K97-0239. In oneembodiment, the compound is PF-1052. In one embodiment, compound isspylidone. In one embodiment, the compound is rubimaillin. In oneembodiment compound inhibits lipid droplet formation.

In one embodiment, the invention provides a method of treating orpreventing viral infection in a mammal, comprising administering to amammalian subject in need thereof a therapeutically effective amount ofan agent that inhibits a long chain fatty acid synthesis enzyme.

The invention also provides a method of treating or preventing viralinfection in a mammal, comprising administering to a mammalian subjectin need thereof a therapeutically effective amount of an agent thatinhibits the activity of a long chain fatty acid synthesis enzyme. Inone embodiment, the enzyme is a long or very long chain acyl-CoAsynthetases. In another embodiment, the enzyme is an elongase. Incertain embodiments of the invention, the enzyme is ACSL1, ELOVL2,ELOVL3, ELOVL6, or SLC27A3. Inhibitors of such enzymes includeinhibitory polynucleotides, small molecule, and biological moleculesincluding, but not limited to, antibodies.

In an embodiment of the invention, the agent is a compound of FormulaVI:

wherein L is selected from carbamate, urea, amide,

wherein R is selected from halo, CF₃, cyclopropyl, optionallysubstituted C₁₋₅ alkyl, wherein the C₁₋₅ alkyl may be substituted withhalo, oxo, —OH, —CN, —NH₂, CO₂H, and C1-3 alkoxy; wherein R₁ is selectedfrom substituted phenyl where the substituents are selected from F, CF₃,Me, OMe, or isopropyl, R₂ is Cl, Ph, 1-(2-pyridone), 4-isoxazol,3-pyrazol, 4-pyrazol, 1-pyrazol, 5-(1,2,4-triazol), 1-(1,2,4-triaol),2-imidazolo, 1-(2-pyrrolidone), 3-(1,3-oxazolidin-2-one); and whereinthe chiral center at C4 can be racemic, (S), (R), or any ratio ofenantiomers, or prodrug thereof, or pharmaceutically acceptable salt orester of said compound or prodrug thereof.

In an embodiment of the invention, the compound of Formula VI isselected from the group consisting of

In an embodiment of the invention, the agent is a compound of FormulaVIIa or VIIb:

wherein R₁ is selected from OMe, OiPr, OCF₃, OPh, CH₂Ph, F, CH₃, CF₃,and benzyl; and R₂ is selected from C₁₋₄ alkyl (such as nBu, nPr, andiPr); phenyl; substituted phenyl where substitutents are selected fromOMe, CF₃, F, tBu, iPr and thio; 2-pyridine; 3-pyridine; and N-methyimidazole, or prodrug thereof, or pharmaceutically acceptable salt orester of said compound or prodrug thereof.

In an embodiment of the invention, the compound of Formula VIIa and VIIbis selected from the group consisting of

In another embodiment the agent is a compound of Formula VIII

wherein R₁ is selected from H, unsubstituted phenyl, substituted phenylwhere substitutents are selected from F, Me, Et, Cl, OMe, OCF₃, and CF₃;C₁₋₆ alkyl (such as Me, Et, iPr, and n-propyl); and C₃₋₆ cycloalkyl(cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl), R₃ and R₄ areindependently selected from H, C₁₋₃ alkyl, and phenyl; or R₃ and R₄taken together form a cycloalkyl of formula —(CH2)_(n)— where n=2, 3, 4and 5, wherein R₅ is selected from methyl; CF₃; cyclopropyl;unsubstituted phenyl; mono- and disubstituted phenyl where substitutentsare selected from F, Me, Et, CN, iPr, Cl, OMe, OPh, OCF₃, and CF₃;unsubstituted heteroaromatic groups (such as 2, 3, or 4-pyridine,isoxazol, pyrazol, triazol); and imidazolo or prodrug thereof, orpharmaceutically acceptable salt or ester of said compound or prodrugthereof.

In an embodiment of the invention, the compound of Formula VIII isselected from the group consisting of

In yet another embodiment, the agent is a compound of Formula IX:

wherein L is selected from urea or an amide, for example

wherein R₁ is selected form 2-, 3-, and 4-pyridine; pyrimidine;unsubstituted heteroaryls such as isoxazol, pyrazol, triazol, imidazole;and unsubstituted phenyl; ortho, meta or para-substituted phenyl wheresubstitutents are F, Me, Et, Cl, OMe, OCF₃, and CF₃, Cl, iPr and phenyl;and wherein R₂ is selected from Cl; iPr; phenyl; ortho, meta orpara-substituted phenyl where substitutents are F, Me, Et, Cl, OMe,OCF₃, and CF₃; and heteroaryls such as 2-, 3-, and 4-pyridine,pyrimidine, and isoxazol, pyrazol, triazol, and imidazo, or a prodrugthereof, or pharmaceutically acceptable salt or ester of said compoundor prodrug thereof.

In one embodiment, the compound of Formula IX is selected from the groupconsisting of

The invention also provides a method of treating or preventing viralinfection in a mammal, comprising administering to a mammalian subjectin need thereof a therapeutically effective amount of an agent thatinhibits acyl-coenzyme A cholesterol acyltransferase (ACAT). In oneembodiment, the agent is a dual inhibitor of acyl-coenzyme A cholesterolacyltransferase 1 (ACAT1) and acyl-coenzyme A cholesterolacyltransferase 2 (ACAT2). In another embodiment, the agent isavasimibe. In yet another embodiment, the agent is rubimaillin.

The invention provides a method of treating or preventing viralinfection in a mammal, comprising administering to a mammalian subjectin need thereof a therapeutically effective amount of an agent thatinhibits ADP-ribosyltransferase 1 (ART1). In one embodiment, the methodcomprises administering to a mammalian subject in need thereof atherapeutically effective amount of meta-iodo-benzylguanidine (MIBG).

The invention further provides a method of treating or preventing viralinfection in a mammal, comprising administering to a mammalian subjectin need thereof a therapeutically effective amount of an agent thatinhibits alanine-glyoxylate aminotransferase 2 (AGXT2). In oneembodiment, the method comprises administering to a mammalian subject inneed thereof a therapeutically effective amount of aminooxyacetic acid(AOAA).

The invention also provides for treatment of viral infections usingcombinations of such agents. In one embodiment, the invention provides amethod of treating or preventing HCMV infection in a mammal, whichcomprises administering to a mammalian subject in need thereof atherapeutically effective amount of an agent that inhibits HCMV-encodedDNA polymerase and an agent that inhibits lipid droplet formation orinhibits an enzyme selected from the group consisting of ACSL1, ELOVL2,ELOVL3, ELOVL6, SLC27A3, FAS, ACC, or HMG-CoA.

In another embodiment, a method of treating or preventing HSV infectionin a mammal is provided, which comprises administering to a mammaliansubject in need thereof a therapeutically effective amount of an agentthat inhibits HSV-encoded DNA polymerase and an agent that inhibitslipid droplet formation or inhibits an enzyme selected from the groupconsisting of ACSL1, ELOVL2, ELOVL3, ELOVL6, SLC27A3, FAS, ACC, orHMG-CoA.

In still another embodiment, the invention provides a method of treatingor preventing influenza infection in a mammal, which comprisesadministering to a mammalian subject in need thereof a therapeuticallyeffective amount of an agent that inhibits influenza-encoded M2 proteinand an agent that inhibits lipid droplet formation or inhibits an enzymeselected from the group consisting of ACSL1, ELOVL2, ELOVL3, ELOVL6,SLC27A3, FAS, ACC, or HMG-CoA.

In yet another embodiment, the invention provides a method of treatingor preventing HCV infection in a mammal, which comprises administeringto a mammalian subject in need thereof a therapeutically effectiveamount of an agent that inhibits HCV RNA synthesis and an agent thatinhibits lipid droplet formation or a cellular long or inhibits anenzyme selected from the group consisting of ACSL1, ELOVL2, ELOVL3,ELOVL6, SLC27A3, FAS, ACC, or HMG-CoA.

The invention also provides a method of treating or preventing HBVinfection in a mammal, which comprises administering to a mammaliansubject in need thereof a therapeutically effective amount of an agentthat inhibits HBV-encoded reverse transcriptase and an agent thatinhibits lipid droplet formation or inhibits an enzyme selected from thegroup consisting of ACSL1, ELOVL2, ELOVL3, ELOVL6, SLC27A3, FAS, ACC, orHMG-CoA.

The invention further provides a method of treating or preventing HIVinfection in a mammal, which comprises administering to a mammaliansubject in need thereof a therapeutically effective amount of an agentthat inhibits HIV-encoded transcriptase and an agent that inhibits lipiddroplet formation or inhibits an enzyme selected from the groupconsisting of ACSL1, ELOVL2, ELOVL3, ELOVL6, SLC27A3, FAS, ACC, orHMG-CoA.

The invention also provides a method for treating or preventing viralinfection in a mammal, comprising administering to a mammalian subjectin need thereof a therapeutically effective amount of an agent thatinhibits the activity of an enzyme selected from ADP-ribosyltransferase1 (ART1), 1-acylglycerol-3-phosphate O-acyltransferase 7 (AGPAT7),alanine-glyoxylate aminotransferase 2 (AGXT2), ADP-ribosyltransferase 3(ART3), leukotriene C4 synthase (LTC4S) transcript variant 2,coactivator-associated arginine methyltransferase 1 (CARM1),chromodomain protein, Y-linked, 2A (CDY2A), FKBP6-like (LOC541473),coagulation factor XIII A1 polypeptide (F13A1), transaldolase 1(TALDO1), gamma-glutamyltransferase 3 (GGT3), heparan sulfate6-O-sulfotransferase 1 (HS6ST1), uronyl-2-sulfotransferase (UST),transketolase-like 1 (TKTL1), phospholipase A2, group VII(platelet-activating factor acetylhydrolase, plasma) (PLA2G7),alanine-glyoxylate aminotransferase 2-like 1 (AGXT2L1), heparan sulfate6-O-sulfotransferase 2 (HS6ST2) transcript variant S,methylcrotonoyl-Coenzyme A carboxylase 2 (beta) (MCCC2), proteindisulfide isomerase family A member 6 (PDIA6), phenylethanolamineN-methyltransferase (PNMT), acetyl-Coenzyme A carboxylase alpha (ACACA)transcript variant 6, UDP glycosyltransferase 3 family polypeptide A2(UGT3A2), glycine amidinotransferase (L-arginine: glycineamidinotransferase) (GATM), glycerol-3-phosphate acyltransferase,mitochondrial (GPAM), microsomal glutathione S-transferase 3 (MGST3),carbonic anhydrase VII (CA7), otopetrin 2 (OTOP2), otopetrin 3 (OTOP3),thromboxane A synthase 1 (TBXAS1), thymidylate synthase (TYMS),thioredoxin domain containing 11 (TXNDC11), protein disulfide isomerasefamily A, member 5 (PDIA5), prostaglandin-endoperoxide synthase 2(PTGS2), syntaxin 6 (STX6), and syntaxin 8 (STX8).

The invention provides a pharmaceutical composition for carrying out theabove methods. The invention also provides a pharmaceutical compositionfor treatment or prevention of a viral infection comprising atherapeutically effective amount of an inhibitor of an enzyme involvedin elongation of very long chain fatty acids and an inhibitor of asecond enzyme that controls virus replication.

The invention provides a method of identifying a compound for treatingor preventing a virus infection, which comprises selecting a compoundthat inhibits a long chain fatty acid synthesis enzyme. According to theinvention, the long chain fatty acid synthesis enzyme can have beenidentified as a regulator of viral replication by treating a test cellinfected with a virus with an agent that inhibits the long chain fattyacid synthesis enzyme, such that virus replication in the treated testcell is reduced as compared to virus replication in an untreated testcell, thus identifying the long chain fatty acid synthesis enzyme as aregulator of viral replication. In an embodiment of the invention, thelong chain fatty acid synthesis enzyme is selected from an acyl-CoAsynthetase long-chain family member, an elongation of very long chainfatty acids enzyme, and solute carrier family 27 (fatty acidtransporter), member 3 (SLC27A3).

The invention also provides a method of identifying a compound fortreating or preventing a viral infection, which comprises selecting acompound that inhibits a long chain fatty acid synthesis enzyme, whereinthe long chain fatty acid synthesis enzyme was identified as a regulatorof viral replication by treating a test cell infected with a virus withan agent that inhibits the long chain fatty acid synthesis enzyme,wherein virus replication in the treated test cell is reduced ascompared to virus replication in an untreated test cell, thusidentifying the long chain fatty acid synthesis enzyme as a regulator ofviral replication. In an embodiment of the invention, the virus isselected from human cytomegalovirus (HCMV), herpes simplex virus-1(HSV-1), influenza A, and hepatitis C virus. The invention furtherprovides agents for treatment or prevention of a viral infection andpharmaceutical compositions thereof that are selected as inhibitors of along chain fatty acid synthesis enzyme.

DESCRIPTION OF THE FIGURES

FIG. 1. Inhibitors of viral replication. The figure depicts genes forwhich inhibition of their expression by siRNA resulted in reduced HCMVreplication. The genes are grouped by function. Specific inhibitors alsoshown to reduce viral replication are also depicted.

FIG. 2. Dose-dependent inhibition of human cytomegalovirus replicationby triacsin C. Top panels: About 90% confluent MRC5 human fibroblastswere treated with the indicated concentrations of triacsin C or thecarrier in which the drug was dissolved (DMSO) in the presence of 10%fetal calf serum and photographed 96 h later. Bottom left panel: About90% confluent MRC5 fibroblasts were either mock infected or infectedwith the AD169 strain of HCMV at a multiplicity of (0.1 pfu/cell). Theuninfected or infected cells received DMSO (solvent for the drug) or thedrug for 96 h before cells were assayed for viability. Bottom rightpanel: About 90% confluent MRC5 fibroblasts were infected with HCMV at amultiplicity of (0.1 pfu/cell) in the presence of DMSO (0 nM triacsin C)or triacsin C at the indicated concentrations. Infected fibroblasts weremaintained in medium containing 10% fetal calf serum. Virus yield in themedium at 96 h after infection was determined by infecting fibrobalstsand assaying IE1 expression by immunofluorescence 24 h later. 96 h afterinfection, cells were harvested into culture medium and sonicated torelease cell-associated virus. Virus yield in the medium was determinedby infecting fibroblasts and assaying IE1 expression byimmunofluorescence 24 h later.

FIG. 3. Dose-dependent inhibition of multiple viruses by triacsin C.About 90% confluent MRC5 fibroblasts were infected with HCMV AD169strain (MOI=0.1 pfu/cell), HSV-1 F-strain (MOI=1 pfu/cell), adenovirustype 5 (MOI=1 pfu/cell) or influenza A strain WSN/33 (MOI=1 pfu/cell).Infected cultures received the indicated doses of drug when the virusinoculum was added, and cultures were maintained in medium plus 10%fetal calf serum plus drug until harvest. Cells were harvested at 96 h(HCMV), 72 h (adenovirus), 48 h (HSV-1) or 24 h post infection(influenza A), and infectious virus was quantified.

FIG. 4. Dose- and time-post-infection-dependent inhibition of HCMV byaminoxyacetic acid. MRC-5 fibroblast cells were infected with HCMV inthe presence of the indicated concentrations of aminoxyacetic acid(AOAA) or the carrier in which the drug was dissolved (PBS) andphotographed at indicated times after infection. Cells were harvested at96 h and virus production was determined assaying IE1 expression in theinfected fibroblasts by immunofluorescence 24 h later.

FIG. 5. Dose- and time-post-infection-dependent inhibition of influenzaA by aminoxyacetic acid. Top panels: Approximately 90% confluent MadinDarby Canine Kidney (MDCK) cells were infected with WSN/33 strain ofInfluenza A virus in the presence of indicated concentrations ofaminoxyacetic acid (AOAA) or the carrier in which the drug was dissolved(PBS) and photographed at indicated times after infection. Bottom panel:Approximately 90% confluent MDCK cells were infected with WSN/33 at amultiplicity of (0.001 pfu/cell) in the presence of PBS (0 mM) or AOAAat the indicated concentrations. Virus yield in the medium at indicatedtimes after infection was determined by standard plaque assay on MDCKcells.

FIG. 6. Dose-dependent inhibition of adenovirus production byaminoxyacetic acid. Top panels: MRC5 fibroblasts were infected withadenovirus in the presence of indicated concentrations of aminoxyaceticacid (AOAA) or the carrier in which the drug was dissolved (PBS) andphotographed at 72 hours after infection. Bottom panel: Approximately90% confluent MRC5 cells were infected with adenovirus at a multiplicityof 1 pfu/cell in the presence of PBS (0 mM AOAA) or AOAA at theindicated concentrations. Virus production at 72 h after infection wasdetermined by standard plaque assay on HeLa cells.

FIG. 7 Dose-dependent inhibition of HCMV production bymeta-iodo-benzylguanidine (MIBG). Replication of HCMV was tested in MRC5fibroblasts. Cells were infected with HCMV and incubated at MIBGconcentrations of 0 μM, 50 μM, 100 μM, and 250 μM. Virus production at96 hours after infection was determined.

FIG. 8 Dose-dependent inhibition of HCMV production by simvastatin.Replication of HCMV was tested in MRC5 fibroblasts. Cells were infectedwith HCMV and incubated at simvastatin concentrations of 0 μM, 1 μM, 2.5μM, and 5 μM. Virus production at 96 hours after infection wasdetermined.

FIG. 9. Dose dependent inhibition of hepatitis C virus by triacsin C.Huh7.5 cells were infected with a derivative of HCV JFH1 strain (MOI=0.1TCID₅₀/cell). Infected cells were maintained in medium containing 10%fetal calf serum plus DMSO (0 nM triacsin C) or triacsin C at theindicated concentrations. Media were harvested at 72 h and infectiousvirus was quantified by standard TCID₅₀ assay on Huh7.5 cells.

FIG. 10. Dose dependent inhibition of HCMV by PF-1052. About 90%confluent MRC5 human fibroblasts were infected with HCMV. Infected cellswere maintained in medium containing 10% fetal calf serum plus theindicated concentrations of PF-1052 or the carrier in which the drug wasdissolved (ethanol). Top panels: The cells were photographed 96 h later.Bottom panel: Virus production at 96 h after infection was determined.

FIG. 11. Long term inhibition of HCMV by PF-1052. About 90% confluentMRC5 human fibroblasts were infected with HCMV. Infected cells weremaintained in medium containing 10% fetal calf serum plus 5 μM PF-1052or the carrier in which the drug was dissolved (ethanol). The media ofthe cells were replaced every day. Starting from 4 days after infection,media were harvested at indicated times and infectious virus wasdetermined. PF-1052 was no longer added to the cells 14 days afterinfection and infectious virus released into media were quantified 2 and5 d after the removal of the drug.

FIG. 12. Dose dependent inhibition of influenza A virus by PF-1052. MRC5cells were infected with influenza A WSN/33 strain (MOI=0.1 pfu/cell).Infected cells were maintained in medium containing 10% fetal calf serumplus the indicated concentrations of PF-1052 or the carrier in which thedrug was dissolved (ethanol). Virus yield in the medium at 24 h afterinfection was determined.

FIG. 13. Dose dependent inhibition of HSV-1 by PF-1052. MRC5 cells wereinfected with HSV-1, KOS strain (MOI=1 pfu/cell). Infected cells weremaintained in medium containing 10% fetal calf serum plus the indicatedconcentrations of PF-1052 or the carrier in which the drug was dissolved(ethanol). Virus yield in the medium at 24 h after infection wasdetermined.

FIG. 14. Adenovirus replication is not inhibited by PF-1052. MRC5 cellswere infected with adenovirus type 5 (MOI=1 pfu/cell). Infected cellswere maintained in medium containing 10% fetal calf serum plus theindicated concentrations of PF-1052 or the carrier in which the drug wasdissolved (ethanol). Virus yield in the medium at 72 h after infectionwas determined.

FIG. 15. Dose dependent inhibition of hepatitis C virus by PF-1052.Huh7.5 cells were infected with a derivative of HCV, JFH1 strain(MOI=0.1 TCID₅₀/cell) in the presence of ethanol (0 μM PF-1052) orPF-1052 at the indicated concentrations. Virus yield in the medium at 72h after infection was determined. The star (*) indicates that the virusyield is below the detection limit which is 1.43E+0.1 TCID₅₀/ml for thisassay.

FIG. 16. Inhibition of HCMV production by rubimaillin. Replication ofHCMV was tested in MRC5 fibroblasts. Cells were infected with HCMV andeither incubated with rubimaillin at concentration of 10 μM or thecarrier in which the drug was dissolved (ethanol). Virus production at96 h after infection was determined.

FIG. 17. Dose dependent inhibition of HCMV by the compound (S)-1y. About90% confluent MRC5 human fibroblasts were infected with HCMV. Infectedcells were maintained in medium containing 10% fetal calf serum plus theindicated concentrations of (S)-1y, Racemic-1y, (R)-1y or the carrier inwhich the drugs were dissolved (DMSO; 0 μM). Top panels: The cells werephotographed 96 h later. Bottom panel: Virus production at 96 h afterinfection was determined.

FIG. 18. Dose dependent inhibition of HCMV by the compound Endo-1k.About 90% confluent MRC5 human fibroblasts were infected with HCMV.Infected cells were maintained in medium containing 10% fetal calf serumplus the indicated concentrations of Endo-1k, Exo-1w, or the carrier inwhich the drugs were dissolved (DMSO; 0 μM). Top panels: The cells werephotographed 96 h later. Bottom panel: Virus production at 96 h afterinfection was determined.

FIG. 19. Dose dependent inhibition of HCMV by the compound 37. About 90%confluent MRC5 human fibroblasts were infected with HCMV. Infected cellswere maintained in medium containing 10% fetal calf serum plus theindicated concentrations of compound 37 or the carrier in which the drugwere dissolved (DMSO; 0 μM). Top panels: The cells were photographed 96h later. Bottom panel: Virus production at 96 h after infection wasdetermined.

FIG. 20. Gene expression analysis of ACSLs during HCMV infection. Upperpanel: MRC5 cells were infected with HCMV AD169 strain (MOI=3 pfu/cell)or remained uninfected (mock). The cells were harvested 48 hours afterinfection and the transcript levels of all five ACSLs were determined byqRT-PCR. The change in transcript levels during infection is shown.Lower panels: MRC5 cells were infected with HCMV AD169 strain (MOI=3pfu/cell) or remained uninfected (M). The cells were harvested atindicated times after infection and processed for western blot. Thelevel of ACSL1 was determined by using an ACSL1-specific monoclonalantibody. β-actin was employed as a loading control.

FIG. 21. Dose dependent inhibition of hepatitis C virus by triacsin C.Huh7.5 cells were infected a derivative of HCV JFH1 strain (MOI=0.1TCID₅₀/cell) Infected cells were maintained in medium containing 10%fetal calf serum plus DMSO (0 μg/ml TOFA) or TOFA at the indicateddoses. Media were harvested at 72 h and infectious virus was quantifiedby standard TCID₅₀ assay on Huh7.5 cells. The star (*) indicates thatthe virus yield is below the detection limit which is 1.43E+0.1TCID₅₀/ml for this assay.

DETAILED DESCRIPTION

Viral replication requires energy and macromolecular precursors derivedfrom the metabolic network of the host cell. Using an integratedapproach to profiling metabolic flux, the inventors discoveredalterations of certain metabolite concentrations and fluxes in responseto viral infection. Details of the profiling methods are described inPCT/US2008/006959, which is incorporated by reference in its entirety.Using this approach, certain enzymes in the various metabolic pathways,especially those which serve as key “switches,” have been discovered tobe useful targets for intervention; i.e., as targets for redirecting themetabolic flux to disadvantage viral replication and restore normalmetabolic flux profiles, thus serving as targets for antiviraltherapies. Enzymes involved in initial steps in a metabolic pathway arepreferred enzyme targets. In addition, enzymes that catalyze“irreversible” reactions or committed steps in metabolic pathways can beadvantageously used as enzyme targets for antiviral therapy.

The subsections below describe in more detail the antiviral compoundsand target enzymes of the invention, screening assays for identifyingand characterizing new antiviral compounds, and methods for their use asantiviral therapeutics to treat and prevent viral infections.

In certain embodiments, compounds of the invention may exist in severaltautomeric forms. Accordingly, the chemical structures depicted hereinencompass all possible tautomeric forms of the illustrated compounds.Compounds of the invention may exist in various hydrated forms.

Definitions of the more commonly recited chemical groups are set forthbelow. Certain variables in classes of compounds disclosed herein reciteother chemical groups. Chemical groups recited herein, but notspecifically defined, have their ordinary meaning as would be known by achemist skilled in the art.

A “C_(1-X) alkyl” group is a saturated straight chain or branchednon-cyclic hydrocarbon having from 1 to x carbon atoms. Representative—(C₁₋₈ alkyls) include -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl,-n-hexyl, -n-heptyl and -n-octyl; while saturated branched alkylsinclude -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl,2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl andthe like. A —(C_(1-X) alkyl) group can be substituted or unsubstituted.

The terms “halogen” and “halo” mean fluorine, chlorine, bromine andiodine.

An “aryl” group is an unsaturated aromatic carbocyclic group of from 6to 14 carbon atoms having a single ring (e.g., phenyl) or multiplecondensed rings (e.g., naphthyl or anthryl). Particular aryls includephenyl, biphenyl, naphthyl and the like. An aryl group can besubstituted or unsubstituted.

A “heteroaryl” group is an aryl ring system having one to fourheteroatoms as ring atoms in a heteroaromatic ring system, wherein theremainder of the atoms are carbon atoms. Suitable heteroatoms includeoxygen, sulfur and nitrogen. In certain embodiments, the heterocyclicring system is monocyclic or bicyclic. Non-limiting examples includearomatic groups selected from the following:

wherein Q is CH2, CH═CH, O, S or NH. Further representative examples ofheteroaryl groups include, but are not limited to, benzofuranyl,benzothienyl, indolyl, benzopyrazolyl, coumarinyl, furanyl,isothiazolyl, imidazolyl, isoxazolyl, thiazolyl, triazolyl, tetrazolyl,thiophenyl, pyrimidinyl, isoquinolinyl, quinolinyl, pyridinyl, pyrrolyl,pyrazolyl, 1H-indolyl, 1H-indazolyl, benzo[d]thiazolyl and pyrazinyl.Heteroaryls can be bonded at any ring atom (i.e., at any carbon atom orheteroatom of the heteroaryl ring) A heteroaryl group can be substitutedor unsubstituted. In one embodiment, the heteroaryl group is a C3-10heteroaryl.

A “cycloalkyl” group is a saturated or unsaturated non-aromaticcarbocyclic ring. Representative cycloalkyl groups include, but are notlimited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl,cyclohexyl, cyclohexenyl, 1,3-cyclohexadienyl, 1,4-cyclohexadienyl,cycloheptyl, 1,3-cycloheptadienyl, 1,3,5-cycloheptatrienyl, cyclooctyl,and cyclooctadienyl. A cycloalkyl group can be substituted orunsubstituted. In one embodiment, the cycloalkyl group is a C3-8cycloalkyl group.

A “heterocycloalkyl” group is a non-aromatic cycloalkyl in which one tofour of the ring carbon atoms are independently replaced with aheteroatom from the group consisting of O, S and N. Representativeexamples of a heterocycloalkyl group include, but are not limited to,morpholinyl, pyrrolyl, pyrrolidinyl, thienyl, furanyl, thiazolyl,imidazolyl, pyrazolyl, triazolyl, piperizinyl, isothiazolyl, isoxazolyl,(1,4)-dioxane, (1,3)-dioxolane, 4,5-dihydro-1H-imidazolyl andtetrazolyl. Heterocycloalkyls can also be bonded at any ring atom (i.e.,at any carbon atom or heteroatom of the Heteroaryl ring). Aheterocycloalkyl group can be substituted or unsubstituted. In oneembodiment, the heterocycloalkyl is a 3-7 membered heterocycloalkyl.

In one embodiment, when groups described herein are said to be“substituted,” they may be substituted with any suitable substituent orsubstituents. Illustrative examples of substituents include those foundin the exemplary compounds and embodiments disclosed herein, as well ashalogen (chloro, iodo, bromo, or fluoro); C₁₋₆ alkyl; C₂₋₆ alkenyl; C₂₋₆alkynyl; hydroxyl; C₁₋₆ alkoxyl; amino; nitro; thiol; thioether; imine;cyano; amido; phosphonato; phosphine; carboxyl; thiocarbonyl; sulfonyl;sulfonamide; ketone; aldehyde; ester; oxygen (═O); haloalkyl (e.g.,trifluoromethyl); carbocyclic cycloalkyl, which may be monocyclic orfused or non-fused polycyclic (e.g., cyclopropyl, cyclobutyl,cyclopentyl, or cyclohexyl), or a heterocycloalkyl, which may bemonocyclic or fused or non-fused polycyclic (e.g., pyrrolidinyl,piperidinyl, piperazinyl, morpholinyl, or thiazinyl); carbocyclic orheterocyclic, monocyclic or fused or non-fused polycyclic aryl (e.g.,phenyl, naphthyl, pyrrolyl, indolyl, furanyl, thiophenyl, imidazolyl,oxazolyl, isoxazolyl, thiazolyl, triazolyl, tetrazolyl, pyrazolyl,pyridinyl, quinolinyl, isoquinolinyl, acridinyl, pyrazinyl, pyridazinyl,pyrimidinyl, benzimidazolyl, benzothiophenyl, or benzofuranyl); amino(primary, secondary, or tertiary); o-lower alkyl; o-aryl, aryl;aryl-lower alkyl; CO₂CH₃; CONH₂; OCH₂CONH₂; NH₂; SO₂NH₂; OCHF₂; CF₃;OCF₃.

As used herein, the term “pharmaceutically acceptable salt(s)” refers toa salt prepared from a pharmaceutically acceptable non-toxic acid orbase including an inorganic acid and base and an organic acid and base.Suitable pharmaceutically acceptable base addition salts of thecompounds include, but are not limited to metallic salts made fromaluminum, calcium, lithium, magnesium, potassium, sodium and zinc ororganic salts made from lysine, N,N′-dibenzylethylenediamine,chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine(N-methylglucamine) and procaine. Suitable non-toxic acids include, butare not limited to, inorganic and organic acids such as acetic, alginic,anthranilic, benzenesulfonic, benzoic, camphorsulfonic, citric,ethenesulfonic, formic, fumaric, furoic, galacturonic, gluconic,glucuronic, glutamic, glycolic, hydrobromic, hydrochloric, isethionic,lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic,pantothenic, phenylacetic, phosphoric, propionic, salicylic, stearic,succinic, sulfanilic, sulfuric, tartaric acid, and p-toluenesulfonicacid. Specific non-toxic acids include hydrochloric, hydrobromic,phosphoric, sulfuric, and methanesulfonic acids. Examples of specificsalts thus include hydrochloride and mesylate salts. Others arewell-known in the art, See for example, Remington's PharmaceuticalSciences, 18th eds., Mack Publishing, Easton Pa. (1990) or Remington:The Science and Practice of Pharmacy, 19th eds., Mack Publishing, EastonPa. (1995).

As used herein and unless otherwise indicated, the term “hydrate” meansa compound, or a salt thereof, that further includes a stoichiometric ornon-stoichiometric amount of water bound by non-covalent intermolecularforces.

As used herein and unless otherwise indicated, the term “solvate” meansa compound, or a salt thereof, that further includes a stoichiometric ornon-stoichiometric amount of a solvent bound by non-covalentintermolecular forces.

As used herein and unless otherwise indicated, the term “prodrug” meansa compound derivative that can hydrolyze, oxidize, or otherwise reactunder biological conditions (in vitro or in vivo) to provide compound.Examples of prodrugs include, but are not limited to, derivatives andmetabolites of a compound that include biohydrolyzable moieties such asbiohydrolyzable amides, biohydrolyzable esters, biohydrolyzablecarbamates, biohydrolyzable carbonates, biohydrolyzable ureides, andbiohydrolyzable phosphate analogues. In certain embodiments, prodrugs ofcompounds with carboxyl functional groups are the lower alkyl esters ofthe carboxylic acid. The carboxylate esters are conveniently formed byesterifying any of the carboxylic acid moieties present on the molecule.Prodrugs can typically be prepared using well-known methods, such asthose described by Burger's Medicinal Chemistry and Drug Discovery 6thed. (Donald J. Abraham ed., 2001, Wiley) and Design and Application ofProdrugs (H. Bundgaard ed., 1985, Harwood Academic Publishers Gmfh).

As used herein and unless otherwise indicated, the term “stereoisomer”or “stereomerically pure” means one stereoisomer of a compound, in thecontext of an organic or inorganic molecule, that is substantially freeof other stereoisomers of that compound. For example, a stereomericallypure compound having one chiral center will be substantially free of theopposite enantiomer of the compound. A stereomerically pure compoundhaving two chiral centers will be substantially free of otherdiastereomers of the compound. A typical stereomerically pure compoundcomprises greater than about 80% by weight of one stereoisomer of thecompound and less than about 20% by weight of other stereoisomers of thecompound, greater than about 90% by weight of one stereoisomer of thecompound and less than about 10% by weight of the other stereoisomers ofthe compound, greater than about 95% by weight of one stereoisomer ofthe compound and less than about 5% by weight of the other stereoisomersof the compound, or greater than about 97% by weight of one stereoisomerof the compound and less than about 3% by weight of the otherstereoisomers of the compound. The compounds can have chiral centers andcan occur as racemates, individual enantiomers or diastereomers, andmixtures thereof. All such isomeric forms are included within theembodiments disclosed herein, including mixtures thereof.

Various compounds contain one or more chiral centers, and can exist asracemic mixtures of enantiomers, mixtures of diastereomers orenantiomerically or optically pure compounds. The use of stereomericallypure forms of such compounds, as well as the use of mixtures of thoseforms are encompassed by the embodiments disclosed herein. For example,mixtures comprising equal or unequal amounts of the enantiomers of aparticular compound may be used in methods and compositions disclosedherein. These isomers may be asymmetrically synthesized or resolvedusing standard techniques such as chiral columns or chiral resolvingagents. See, e.g., Jacques, J., et al., Enantiomers, Racemates andResolutions (Wiley-Interscience, New York, 1981); Wilen, S. H., et al.,Tetrahedron 33:2725 (1977); Eliel, E. L., Stereochemistry of CarbonCompounds (McGraw-Hill, N Y, 1962); and Wilen, S. H., Tables ofResolving Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ.of Notre Dame Press, Notre Dame, Ind., 1972).

It should also be noted that compounds, in the context of organic andinorganic molecules, can include E and Z isomers, or a mixture thereof,and cis and trans isomers or a mixture thereof. In certain embodiments,compounds are isolated as either the E or Z isomer. In otherembodiments, compounds are a mixture of the E and Z isomers.

As used herein, the term “effective amount” in the context ofadministering a therapy to a subject refers to the amount of a therapywhich is sufficient to achieve one, two, three, four, or more of thefollowing effects: (i) reduce or ameliorate the severity of a viralinfection or a symptom associated therewith; (ii) reduce the duration ofa viral infection or a symptom associated therewith; (iii) prevent theprogression of a viral infection or a symptom associated therewith; (iv)cause regression of a viral infection or a symptom associated therewith;(v) prevent the development or onset of a viral infection or a symptomassociated therewith; (vi) prevent the recurrence of a viral infectionor a symptom associated therewith; (vii) reduce or prevent the spread ofa virus from one cell to another cell, or one tissue to another tissue;(ix) prevent or reduce the spread of a virus from one subject to anothersubject; (x) reduce organ failure associated with a viral infection;(xi) reduce hospitalization of a subject; (xii) reduce hospitalizationlength; (xiii) increase the survival of a subject with a viralinfection; (xiv) eliminate a virus infection; and/or (xv) enhance orimprove the prophylactic or therapeutic effect(s) of another therapy.

As used herein, the term “effective amount” in the context of a compoundfor use in cell culture-related products refers to an amount of acompound which is sufficient to reduce the viral titer in cell cultureor prevent the replication of a virus in cell culture.

A preferred dose of a triacsin used to treat or prevent viral infectionsin mammals is <100 mg/kg, <50 mg/kg, <20 mg/kg, <10 mg/kg, <5 mg/kg, <2mg/kg, <1 mg/kg, <0.5 mg/kg, <0.2 mg/kg, <0.1 mg/kg, <0.05 mg/kg, <0.02mg/kg, or <0.01 mg/kg. A preferred dose of a triacsin used to treat orprevent viral infections in mammals results in total serumconcentrations of <100 μM, <50 μM, <20 μM, <10 μM, <5 μM, <1 μM, <500nM, or <250 nM. It is noted that higher triacsin C concentrations,e.g., >5 μM, >10 μM, >20 μM, >50 μM, or >100 μM, increase the risk ofside effects including vasodilation (another effect of triacsin C).

The present invention also provides for the use of Triacsin compounds incell culture-related products in which it is desirable to have antiviralactivity. In one embodiment, a triacsin compound is added to cellculture media. The triacsin compounds used in cell culture media includecompounds that may otherwise be found too toxic for treatment of asubject.

1. Host Cell Target Enzymes

The invention further provides cellular target enzymes for reducingvirus production. As described below, an siRNA screen was performed totest the effects of inhibiting specific enzymes on the infectious yieldof HCMV. The siRNA which were found to inhibit HCMV replication areshown in Table 1. Accordingly, the present invention provides methods oftreating viral infections using inhibitors or other modulators of theseenzymes. Inhibitors and modulators include without limitation smallmolecules, nucleic acids, and proteins. As used herein, “small molecule”refers to a substances that has a molecular weight up to 2000 atomicmass units (Daltons). Exemplary nucleic acid-based inhibitors includesiRNA and shRNA. Exemplary protein-based inhibitors include antibodies.Additional small molecule inhibitors can be found by screening ofcompound libraries and/or design of molecules that bind to specificpockets in the structures of these enzymes. The properties of thesemolecules can be optimized through derivitization, including iterativerounds of synthesis and experimental testing.

TABLE 1 Enzyme knockdown by siRNA inhibits HCMV replication Foldreduction Enzyme 41.43 ADP-ribosyltransferase 1 (ART1) 14.701-acylglycerol-3-phosphate O-acyltransferase 7 (AGPAT7) 10.73alanine-glyoxylate aminotransferase 2 (AGXT2) 10.46 solute carrierfamily 27 (fatty acid transporter), member 3 (SLC27A3)  9.83ADP-ribosyltransferase 3 (ART3)  3.45 leukotriene C4 synthase (LTC4S),transcript variant 2  3.22 coactivator-associated argininemethyltransferase 1 (CARM1)  3.06 chromodomain protein, Y-linked, 2A(CDY2A)  2.72 FKBP6-like (LOC541473)  2.63 carbonic anhydrase VII (CA7) 2.63 otopetrin 3 (OTOP3)  2.62 coagulation factor XIII, A1 polypeptide(F13A1)  2.57 acyl-CoA synthetase long-chain family member 1 (ACSL1) 2.56 thromboxane A synthase 1 (TBXAS1)  2.56 thymidylate synthase(TYMS)  2.53 transaldolase 1 (TALDO1)  2.51 gamma-glutamyltransferase 3(GGT3)  2.49 heparan sulfate 6-O-sulfotransferase 1 (HS6ST1)  2.46uronyl-2-sulfotransferase (UST)  2.39 transketolase-like 1 (TKTL1)  2.33phospholipase A2, group VII (platelet-activating factor acetylhydrolase,plasma) (PLA2G7)  2.27 thioredoxin domain containing 11 (TXNDC11)  2.25alanine-glyoxylate aminotransferase 2-like 1 (AGXT2L1)  2.25 heparansulfate 6-O-sulfotransferase 2 (HS6ST2), transcript variant S  2.23methylcrotonoyl-Coenzyme A carboxylase 2 (beta) (MCCC2)  2.23 elongationof very long chain fatty acids (ELOVL2)  2.22 protein disulfideisomerase family A, member 5 (PDIA5)  2.10 protein disulfide isomerasefamily A, member 6 (PDIA6)  2.06 elongation of very long chain fattyacids (ELOVL3)  2.05 ELOVL family member 6, elongation of long chainfatty acids (ELOVL6)  2.04 phenylethanolamine N-methyltransferase (PNMT) 2.02 acetyl-Coenzyme A carboxylase alpha (ACACA), transcript variant 6 2.01 UDP glycosyltransferase 3 family, polypeptide A2 (UGT3A2)  1.97glycine amidinotransferase (L-arginine:glycine amidinotransferase)(GATM)  1.96 prostaglandin-endoperoxide synthase 2 (PTGS2)  1.96syntaxin 8 (STX8)  1.96 glycerol-3-phosphate acyltransferase,mitochondrial (GPAM)  1.94 microsomal glutathione S-transferase 3(MGST3)  1.92 otopetrin 2 (OTOP2)  1.92 syntaxin 6 (STX6)

As depicted in FIG. 1, several of the above enzymes have relatedfunctions. For example, acyl-CoA synthetase long chain family member 1(ACSL1), elongation of very long chain fatty acids 2, 3 and 6 (ELOVL2,ELOVL3 and ELOVL6), and solute carrier family 27 (fatty acidtransporter) member 3 (SLC27A3) (which despite its name has long chainand very long chain acyl-CoA ligase activity) all are enzymes involvedin synthesis of long and very long chain acyl-CoA species. Theobservation that knockdown of any of these enzymes inhibited theproduction of infectious HCMV leads to the conclusion that HCMV requiresacyl-CoA derivatives of long chain fatty acids and very long chain fattyacids for the efficient production of progeny virus.

Accordingly, the invention provides two classes of enzymes (long andvery long chain acyl-CoA synthetases and elongases) as antiviraltargets, including, but not limited to ACSL1, ELOVL2, ELOVL3, ELOVL6,and SLC27A3. Long-chain acyl-CoA synthetases (ACSLs) (E.C.6.2.1.3)catalyze esterification of long-chain fatty acids, mediating thepartitioning of fatty acids in mammalian cells. ACSL isoforms (ACSL1,ACSL3, ACSL4, ACSL5, and ACSL6) generate bioactive fatty acyl-CoAs fromCoA, ATP, and long-chain (C12-C20) fatty acids. In many instances, theenzymes are tissue specific and/or substrate specific. For example,ACSLs exhibit different tissue distribution, subcellular localization,fatty acid preference, and transcriptional regulation. Similarly, sevendistinct fatty acid condensing enzymes (elongases) have been identifiedin mouse, rat, and human, with different substrate specificities andexpression patterns. ELOVL-1, ELOVL-3, and ELOVL-6 elongate saturatedand monounsaturated fatty acids, whereas ELOVL-2, ELOVL-4, and ELOVL-5elongate polyunsaturated fatty acids. ELOVL-5 also elongates somemonounsaturated fatty acids, like palmitoleic acid and specificallyelongates γ-linolenoyl-CoA (18:3,n-6 CoA). ELOVL-2 specificallyelongates 22-carbon PUFA. Also, the elongases (ELOVL) are expresseddifferentially in mammalian tissues. For example, five elongases areexpressed in rat and mouse liver, including ELOVL-1, -2, -3, -5, -6. Incontrast, the heart expresses ELOVL-1, -5, and -6, but not ELOVL-2.

Another means of inhibiting virus production is by targeting a cellularcomponent with a small molecule or other inhibitor of enzyme function.For example, long and very long chain acyl-CoA synthetases can betargeted with triacsin C and its relatives, derivatives, and analogues.As discussed herein, nanomolar concentrations of triacsin C inhibit thereplication of HCMV, herpes simplex virus-1 (HSV-1), and influenza A.

Another set of related enzymes are leukotriene C4 synthase (LTC4S),gamma-glutamyltransferase 3 (GGT3), and microsomalglutathione-S-transferase 3 (MGST3). These enzymes each contribute tothe synthesis of cysteinyl leukotrienes, with LTC4S being the pivotalenzyme. The observation that knockdown of any of these enzymes inhibitedthe production of infectious HCMV leads to the conclusion that HCMVrequires cysteinyl leukotrienes for the efficient production of progenyvirus, and identifies enzymes of cysteinyl leukotriene synthesis asantiviral targets. In addition to siRNA, another inhibitor of cysteinylleukotriene synthesis is caffeic acid. Synthesis of the cysteinylleukotriene precursor leukotriene A4 can be inhibited with zileuton, and100 μM zileuton reduced HCMV replication by ˜90% without evidence ofhost cell toxicity. According to the invention, antiviral agents alsoinclude inhibitors of leukotriene and cysteinyl leukotriene signaling,such as, but not limited to zafirlukast or montelukast.

A pair of related enzymes that are both required for HCMV replicationare ADP-ribosyltransferase 1 and 3 (ART1 and ART3). Inhibition of eitherof these enzymes led to a marked reduction in HCMV replication, ˜40-foldfor ART1 and ˜10-fold for ART3. Without being bound by any particularmechanism, although ADP-ribosyltransfer is not per se a reaction oflipid metabolism, ADP ribosylation plays a key role in regulating lipidstorage via targets including the protein CtBP1/BARS. Mono-ADPribosylation of this protein results in loss of lipid droplets due to adramatic efflux of fatty acids. Monitoring lipid droplets via microscopywith oil red O staining demonstrates that HCMV infection resultsinitially in accumulation of lipid droplets in the infected hosts, andthereafter (by 72 hours post infection) in a dramatic depletion of lipiddroplets. Accordingly, ADP-ribosylation appears to play a key role inregulating these lipid storage events during HCMV infection, and siRNAdata indicates that such regulation is essential for HCMV replication.The observation that knockdown of either of these enzymes inhibited thatproduction of infectious HCMV suggests that HCMV requiresADP-ribosyltransfer activity for efficient production of progeny virus.In addition to siRNA, another means of inhibiting ADP-ribosyltransferaseis with the compound meta-iodobenzylguanidine (MIBG), and 100 μM MIBGinhibited the replication of HCMV in fibroblasts by 13-fold with noevidence of host cell toxicity.

The observations of lipid droplet accumulation and depletion during HCMVinfection in an ordered temporal manner indicates that HCMV hijacks thehost cell machinery involved in lipid droplet production andconsumption. Thus host cell components involved in lipid dropletproduction and consumption provide antiviral targets. In addition tosiRNA against the relevant cellular machinery, other means of inhibitinglipid droplet formation include the compounds spylidone, PF-1052 (afungal natural product isolated from Phoma species), vermisporin,beauveriolides, phenochalasins, isobisvertinol, K97-0239, andrubimaillin. PF-1052 (10 μM) profoundly inhibited HCMV late proteinsynthesis (>99%) and similarly profoundly inhibits HMCV replication. Inaddition, triacsin C also resulted in depletion of lipid droplets, with100 nM triacsin C causing >90% depletion of lipid droplets in HCMVinfected cells and 250 nM resulting in no detectable lipid droplets byoil red 0 staining Normally patterns of HCMV-induced accumulation anddepletion of lipid droplets were also blocked by 100 μM MIBG.

The loss of lipid droplets in HCMV infected cells is followed by theinduction of lipid droplet formation in the neighboring uninfectedcells. This indicates that HCMV infection results in the enhanced uptakeor synthesis of lipids in the surrounding cells. Note that, HCMV spreadoccurs mainly from cell to cell in vivo and lipid accumulation inuninfected cells next to the infected cells can be considered as afacilitating event for the secondary infections. Triacsin C resulted indepletion of lipid droplets both in HCMV infected and surroundinguninfected cells with 100 nM triacsin C causing >90% depletion of lipiddroplets and 250 nM resulting in no detectable lipid droplets by oil redO staining.

The major constituents of lipid droplets are CEs and TGs (estimatedpercentages in macrophages are ˜58 and ˜27 w/w respectively). Among thecompounds indicated above, PF-1052 inhibits both CE and TG synthesis ina dose dependent manner, whereas, rubimaillin (also referred asmollugin) selectively inhibits CE synthesis. Rubimaillin is anaphthohydroquinone isolated from the plant Rubia Cordifoila. Theinhibitory effect of rubimaillin on CE synthesis and lipid dropletformation is linked to its activity on acyl-CoA:cholesterolacyl-transferases (ACATs). It is a dual inhibitor of ACAT1 and ACAT2enzymes (Matsuda et al., 2009, Biol. Pharm. Bull., 32, 1317-1320) and 10μM of rubimaillin reduced HCMV replication by >80%. Thus targeting ACATenzymes, which leads to the inhibition of lipid droplet formation, canbe used in treating virus infections. The examples of dual ACATinhibitors include the compounds pactimibe and avasimibe.

Another pair of related enzymes that are both required for HCMVreplication are alanine-glyoxylate aminotransferase 2 (AGXT2) andalanine-glyoxylate aminotransferase 2-like 1 (AGXT2L1), with knockdownof AGXT2 having a particularly strong impact on viral replication.Without being bound by any particular mechanism, althoughalanine-glyoxylate aminotransferase is not a reaction of lipidmetabolism per se, a major route of glyoxylate production in mammals isduring lipid degradation. Accordingly, the antiviral effects ofknockdown of AGXT2 and AGXT2L1 may arise from HCMV triggering excessiveglyoxylate production which is highly reactive and toxic in biologicalsystems from pathways including lipid degradation, and from thisglyoxylate needing to be converted to glycine and pyruvate for viralreplication to proceed normally. The observation that knockdown ofeither of these enzymes inhibits production of infectious HCMV indicatesthat glyoxylate degradation and/or glycine synthesis activity isrequired for efficient production of progeny virus and identifiesalanine-glyoxylate aminotransferases as antiviral targets. In additionto siRNA, another means of inhibiting alanine-glyoxylateaminotransferase activity, which also impacts other aminotransferases,is via the compound aminooxyacetic acid (AOAA). AOAA inhibited thereplication of each of three different viruses tested: HCMV, influenzaA, and adenovirus. See Example 2.

Yet another pair of related enzymes are transaldolase 1 (TALDO1) andtransketolase-like 1 (TKTL1). Although not catalyzing reactions of lipidmetabolism per se, and without being bound by any particular mechanism,these enzymes both sit in the pentose phosphate pathway, which has amongits major functions production of NADPH, which is used substantially forfatty acid biosynthesis. Another function of the pentose phosphatepathway which may be important for viral replication isribose-5-phosphate synthesis. The observation that knockdown of eitherof these enzymes inhibited that production of infectious HCMV indicatesthat HCMV requires pentose phosphate pathway activity for efficientproduction of progeny virus. Accordingly, antiviral targets includetransaldolase, transketolase, and transketolase-like enzymes.

Fatty acid elongation requires the condensation between fatty acyl-CoAand malonyl-CoA to generate β-ketoacyl-CoA which is the rate limitingstep for the synthesis of long and very long chain fatty acids. Thisstep is catalyzed by ELOVL enzymes and requires a fatty-acyl-CoA as aprecursor, which is generated by ACSLs, and malonyl-CoA, which isproduced by acetyl-coA carboxylase alpha (ACACA; also referred as ACC1).Therefore, in addition to ELOVLs and ACSLs, inhibition of ACACA alsoprovides another means of inhibiting virus production. Consistently,ACACA is identified as an enzyme required for HCMV replication by thesiRNA screen. In addition to siRNA, another means of inhibitingaacetyl-CoA-carboxylase activity, is via the compound TOFA. TOFAinhibited the replication of each of the two different viruses: HCMV andHCV (see Example 11).

An enzyme which is required for HCMV replication is carbonic anhydrase 7(CA7). Although not catalyzing the reactions of lipid metabolism per se,this enzyme catalysis the hydration of carbon dioxide to producebicarbonate which is substantially required for the synthesis ofmalonyl-CoA from acetyl-coA, which is the rate limiting step of fattyacid biosynthesis. Carbonic anhydrases can be inhibited byacetazolamide, and 25 μM acetazolamide inhibited HCMV replication by˜80% without evidence of host cell cytotoxicity.

2. Targeting Combinations of Host Cell Enzymes

Lipid-related processes are essential to viral growth, replicationand/or other elements of infection. Consequently, it is likely thatmultiple cellular enzymes that function in lipid metabolism are neededfor successful infection, and it is possible that simultaneousinhibition of multiple enzymes (e.g., two or more different enzymes)will produce a synergistic inhibition of infection or allow the use oflower doses of each compound to achieve a desirable therapeutic effect.Accordingly, the present invention relates to the prevention andtreatment of viral infection in a mammal in need thereof, viaadministering to the mammal two or more compounds described herein,wherein each compound targets one or more different enzymes describedherein. In some embodiments, such combination therapy is sequential; inother embodiments, it is simultaneous. In some embodiments, the two ormore agents are formulated together to create a composition comprisingtwo or more compounds for the prevention and/or treatment of viralinfection via modulation of host cell lipid and/or cholesterolmetabolism. In some embodiments, the dose of one of the compounds issubstantially less, e.g., 1.5, 2, 3, 5, 7, or 10-fold less, thanrequired when used independently for the prevention and/or treatment ofviral infection. In some embodiments, the dose of both agents is reducedby 1.5, 2, 3, 5, 7, or 10-fold or more.

As used herein, the term “in combination,” in the context of theadministration of two or more therapies to a subject, refers to the useof more than one therapy (e.g., more than one prophylactic agent and/ortherapeutic agent). The use of the term “in combination” does notrestrict the order in which therapies are administered to a subject witha viral infection. A first therapy (e.g., a first prophylactic ortherapeutic agent) can be administered prior to (e.g., 5 minutes, 15minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks,4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantlywith, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6weeks, 8 weeks, or 12 weeks after) the administration of a secondtherapy to a subject with a viral infection.

In one embodiment, a triacsin compound or relative or analog is combinedwith an inhibitor of a long or very long chain fatty acid synthesisenzyme, including, but not limited to, an inhibitor of ACSL1, ELOVL2,ELOVL6, or SLC27A3. In another embodiment, the triacsin compound orrelative or analog is combined with an inhibitor of HMG-CoA reductase.In another embodiment, the triacsin compound or relative or analog iscombined with an inhibitor of acetyl-CoA carboxylase. In still anotherembodiment, the triacsin compound or relative or analog is combined withan inhibitor of a fatty acid synthase. In certain embodiments of theinvention, the triacsin compound is triacsin C.

In certain embodiments of the invention, combinations include one ormore drugs from group A and one or more drugs from group B, whereingroup A comprises inhibitors of the HCMV-encoded DNA polymeraseincluding, but not limited to, gancyclovir, valgancyclovir, cidofovir,and foscarnet, and group B compromises inhibitors of cellular long andvery long chain fatty acid metabolic enzymes and processes including,but not limited to, ACSL1, ELOVL2, ELOVL3, ELOVL6, FAS, SLC27A3, ACC,HMG-CoA reductase, and lipid droplet formation, for the treatment ofpathological manifestations of HCMV infection, such as retinitis,colitis, hepatitis, pneumonitis, esophagitis, polyradiculopathy,transverse myelitis, subacute encephalitis, mononucleosis and congenitalHCMV infection.

In another combination, group A comprises inhibitors of the HSV-encodedDNA polymerase including, but not limited to, acyclovir, valacyclovir,pencyclovir, and famcyclovir, and group B compromises inhibitors ofcellular long and very long chain fatty acid metabolic enzymes andprocesses including, but not limited to, ACSL1, ELOVL2, ELOVL3, ELOVL6,FAS, SLC27A3, ACC, HMG-CoA reductase, and lipid droplet formation, forthe treatment of pathological manifestations of HSV-1 or HSV-2infection, such as herpes labialis, herpes genitalis, encephalitis,meningitis, esophagitis, herpetic gingivostomatitis, herpetickeratoconjunctivitis, herpetic sycosis, eczema herpeticum and congenitalherpes simplex infection.

In another combination, group A comprises inhibitors of theinfluenza-encoded M2 protein including, but not limited to, amantadineand rimantadine, and inhibitors of the influenza-encoded neuraminidase,including, but not limited to, oseltamivir, peramivir and zanamivir, andgroup B compromises inhibitors of cellular long and very long chainfatty acid metabolic enzymes and processes including, but not limitedto, ACSL1, ELOVL2, ELOVL3, ELOVL6, FAS, SLC27A3, ACC, HMG-CoA reductase,and lipid droplet formation, for the treatment of pathologicalinfections of influenza A, influenza B or influenza C.

In another combination, group A comprises the HCV RNA synthesisinhibitor ribavirin, and the immunomodulators peginterferon alfa-2a(PEGASYS™) and peginterferon alfa-2b (PEG-INTRON™), and group Bcompromises inhibitors of cellular long and very long chain fatty acidmetabolic enzymes and processes including, but not limited to, ACSL1,ELOVL2, ELOVL3, ELOVL6, FAS, SLC27A3, ACC, HMG-CoA reductase, and lipiddroplet formation, for the treatment of either hepatitis C or chronic,asymptomatic HCV infection.

In another combination, group A comprises the nucleoside or nucleotideinhibitors of HBV reverse transcriptase, including, but not limited to,lamivudine, adefovir, tenofovir, telbivudine, and entecavir, and theimmunomodulators peginterferon alfa-2a (PEGASYS™) and peginterferonalfa-2b (PEG-INTRON™), and group B compromises inhibitors of cellularlong and very long chain fatty acid metabolic enzymes and processesincluding, but not limited to, ACSL1, ELOVL2, ELOVL3, ELOVL6, FAS,SLC27A3, ACC, HMG-CoA reductase, and lipid droplet formation, for thetreatment of either hepatitis B or chronic, asymptomatic HBV infection.

In another combination, group A comprises nucleoside or nucleotideanalog inhibitors of the HIV-encoded reverse transcriptase including,but not limited to, abacavir, didanosine, emtricitabine, lamivudine,stavudine, tenofovir and zidovudine; non-nucleotide analog inhibitors ofthe HIV-encoded reverse transcriptase including, but not limited to,delavirdine, efavirenz, etravirine and nevirapine; inhibitors of theHIV-encoded protease, including, but not limited to, amprenavir,atazanavir, darunavir, fosamprenavir, indinavir, lopinavir, ritonavir,nelfinavir, saquinavir and tipranavir; inhibitors of the HIV-encodedintegrase, including, but not limited to, raltegravir; or inhibitors ofHIV entry, including, but not limited to, enfuvirtide and maraviroc, andgroup B compromises inhibitors of cellular long and very long chainfatty acid metabolic enzymes and processes including, but not limitedto, ACSL1, ELOVL2, ELOVL3, ELOVL6, FAS, SLC27A3, ACC, HMG-CoA reductase,and lipid droplet formation, for the treatment of pathologicalmanifestations of HIV-1 or HIV-2 infection, such as acquiredimmunodeficiency syndrome (AIDS). Of particular interest arecombinations between drugs of group B and the group A combinationtherapies currently recommended for Highly Active Antiretroviral Therapy(HAART), which include efavirenz/emtricitabine/tenofovir (ATRIPLA™);emtricitabine/tenofovir (TRUVADA™) plus raltegravir (ISENTRESS™);darunavir (PREZISTA™) plus ritonavir (NORVIR™) andemtricitabine/tenofovir (TRUVADA™); and atazanavir (REYATAZ™) plusritonavir (NORVIR™) and emtricitabine/tenofovir (TRUVADA™).

In certain embodiments of the invention, an inhibitor of cellular longand very long chain fatty acid metabolic enzymes and processesincluding, but not limited to, ACSL1, ELOVL2, ELOVL3, ELOVL6, FAS,SLC27A3, ACC, HMG-CoA reductase, and lipid droplet formation, is used incombination with another such inhibitor for the treatment or preventionof viral infection including but not limited to infection by HCMV,HSV-1, HSV-2, influenza A, influenza B, influenza C, HCV, HBV, HIV-1 orHIV-2. Non-limiting examples of such combinations include an ACSL1inhibitor and an ELOVL2 inhibitor, an ACSL1 inhibitor and an ELOVL3inhibitor, an ACSL1 inhibitor and an ELOVL6 inhibitor, an ACSL1inhibitor and an SLC27A3 inhibitor, an ACSL1 inhibitor and an ACCinhibitor, an ACSL1 inhibitor and an FAS inhibitor, an ACSL1 inhibitorand an HMG-CoA reductase inhibitor. Further examples include an ELOVL2inhibitor and an SLC27A3 inhibitor, an ELOVL2 inhibitor and an ACCinhibitor, an ELOVL2 inhibitor and an FAS inhibitor, an ELOVL2 inhibitorand an HMG-CoA reductase inhibitor, an ELOVL3 inhibitor and an SLC27A3inhibitor, an ELOVL3 inhibitor and an ACC inhibitor, an ELOVL3 inhibitorand an FAS inhibitor, an ELOVL3 inhibitor and an HMG-CoA reductaseinhibitor, an ELOVL6 inhibitor and an SLC27A3 inhibitor, an ELOVL6inhibitor and an ACC inhibitor, an ELOVL6 inhibitor and an FASinhibitor, an ELOVL6 inhibitor and an HMG-CoA reductase inhibitor, anSLC27A3 inhibitor and an ACC inhibitor, an SLC27A3 inhibitor and an FASinhibitor, an SLC27A3 inhibitor and an HMG-CoA reductase inhibitor, anELOVL2 inhibitor and an ELOVL3 inhibitor, an ELOVL2 inhibitor and anELOVL 6 inhibitor, and an ELOVL3 inhibitor and an ELOVL6 inhibitor.

2.1 Inhibitors of Host Cell Enzymes

2.1.1 RNAi Molecules

According to the invention, RNA interference is used to reduceexpression of a target enzyme in a cell in order to reduce yield ofinfectious virus. siRNAs were designed to inhibit expression of avariety of enzyme targets. 30 targets for which infection yields of HCMVwere reduced by the an siRNA, along with descriptions of the interferingRNA, are provided below in Example 1. In certain embodiments, a compoundis an RNA interference (RNAi) molecule that can decrease the expressionlevel of a target enzyme. RNAi molecules include, but are not limitedto, small-interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA(miRNA), and any molecule capable of mediating sequence-specific RNAi.

RNA interference (RNAi) is a sequence specific post-transcriptional genesilencing mechanism triggered by double-stranded RNA (dsRNA) that havehomologous sequences to the target mRNA. RNAi is also calledpost-transcriptional gene silencing or PTGS. See, e.g., Couzin, 2002,Science 298:2296-2297; McManus et al., 2002, Nat. Rev. Genet. 3,737-747; Hannon, G. J., 2002, Nature 418, 244-251; Paddison et al.,2002, Cancer Cell 2, 17-23. dsRNA is recognized and targeted forcleavage by an RNaseIII-type enzyme termed Dicer. The Dicer enzyme“dices” the RNA into short duplexes of about 21 to 23 nucleotides,termed siRNAs or short-interfering RNAs (siRNAs), composed of 19nucleotides of perfectly paired ribonucleotides with about two threeunpaired nucleotides on the 3′ end of each strand. These short duplexesassociate with a multiprotein complex termed RISC, and direct thiscomplex to mRNA transcripts with sequence similarity to the siRNA. As aresult, nucleases present in the RNA-induced silencing complex (RISC)cleave and degrade the target mRNA transcript, thereby abolishingexpression of the gene product.

Numerous reports in the literature purport the specificity of siRNAs,suggesting a requirement for near-perfect identity with the siRNAsequence (Elbashir et al., 2001. EMBO J. 20:6877-6888; Tuschl et al.,1999, Genes Dev. 13:3191-3197; Hutvagner et al., Sciencexpress297:2056-2060). One report suggests that perfect sequencecomplementarity is required for siRNA-targeted transcript cleavage,while partial complementarity will lead to translational repressionwithout transcript degradation, in the manner of microRNAs (Hutvagner etal., Sciencexpress 297:2056-2060).

miRNAs are regulatory RNAs expressed from the genome, and are processedfrom precursor stem-loop (short hairpin) structures (approximately 80nucleotide in length) to produce single-stranded nucleic acids(approximately 22 nucleotide in length) that bind (or hybridizes) tocomplementary sequences in the 3′ UTR of the target mRNA (Lee et al.,1993, Cell 75:843-854; Reinhart et al., 2000, Nature 403:901-906; Lee etal., 2001, Science 294:862-864; Lau et al., 2001, Science 294:858-862;Hutvagner et al., 2001, Science 293:834-838). miRNAs bind to transcriptsequences with only partial complementarity (Zeng et al., 2002, Molec.Cell 9:1327-1333) and repress translation without affecting steady-stateRNA levels (Lee et al., 1993, Cell 75:843-854; Wightman et al., 1993,Cell 75:855-862). Both miRNAs and siRNAs are processed by Dicer andassociate with components of the RNA-induced silencing complex(Hutvagner et al., 2001, Science 293:834-838; Grishok et al., 2001, Cell106: 23-34; Ketting et al., 2001, Genes Dev. 15:2654-2659; Williams etal., 2002, Proc. Natl. Acad. Sci. USA 99:6889-6894; Hammond et al.,2001, Science 293:1146-1150; Mourlatos et al., 2002, Genes Dev.16:720-728).

Short hairpin RNA (shRNA) is a single-stranded RNA molecule comprisingat least two complementary portions hybridized or capable of hybridizingto form a double-stranded (duplex) structure sufficiently long tomediate RNAi upon processing into double-stranded RNA with overhangs,e.g., siRNAs and miRNAs. shRNA also contains at least onenoncomplementary portion that forms a loop structure upon hybridizationof the complementary portions to form the double-stranded structure.shRNAs serve as precursors of miRNAs and siRNAs.

Usually, sequence encoding an shRNA is cloned into a vector and thevector is introduced into a cell and transcribed by the cell'stranscription machinery (Chen et al., 2003, Biochem Biophys Res Commun311:398-404). The shRNAs can then be transcribed, for example, by RNApolymerase III (Pol III) in response to a Pol III-type promoter in thevector (Yuan et al., 2006, Mol Biol Rep 33:33-41 and Scherer et al.,2004, Mol Ther 10:597-603). The expressed shRNAs are then exported intothe cytoplasm where they are processed by proteins such as Dicer intosiRNAs, which then trigger RNAi (Amarzguioui et al., 2005, FEBS Letter579:5974-5981). It has been reported that purines are required at the 5′end of a newly initiated RNA for optimal RNA polymerase IIItranscription. More detailed discussion can be found in Zecherle et al.,1996, Mol. Cell. Biol. 16:5801-5810; Fruscoloni et al., 1995, NucleicAcids Res, 23:2914-2918; and Mattaj et al., 1988, Cell, 55:435-442. TheshRNAs core sequences can be expressed stably in cells, allowinglong-term gene silencing in cells both in vitro and in vivo, e.g., inanimals (see, McCaffrey et al., 2002, Nature 418:38-39; Xia et al.,2002, Nat. Biotech. 20:1006-1010; Lewis et al., 2002, Nat. Genetics32:107-108; Rubinson et al., 2003, Nat. Genetics 33:401-406; andTiscornia et al., 2003, Proc. Natl. Acad. Sci. USA 100:1844-1848).

Martinez et al. reported that RNA interference can be used toselectively target oncogenic mutations (Martinez et al., 2002, Proc.Natl. Acad. Sci. USA 99:14849-14854). In this report, an siRNA thattargets the region of the R248W mutant of p53 containing the pointmutation was shown to silence the expression of the mutant p53 but notthe wild-type p53.

Wilda et al. reported that an siRNA targeting the M-BCR/ABL fusion mRNAcan be used to deplete the M-BCR/ABL mRNA and the M-BCR/ABL oncoproteinin leukemic cells (Wilda et al., 2002, Oncogene 21:5716-5724).

U.S. Pat. No. 6,506,559 discloses a RNA interference process forinhibiting expression of a target gene in a cell. The process comprisesintroducing partially or fully doubled-stranded RNA having a sequence inthe duplex region that is identical to a sequence in the target geneinto the cell or into the extracellular environment.

U.S. Patent Application Publication No. US 2002/0086356 discloses RNAinterference in a Drosophila in vitro system using RNA segments 21-23nucleotides (nt) in length. The patent application publication teachesthat when these 21-23 nt fragments are purified and added back toDrosophila extracts, they mediate sequence-specific RNA interference inthe absence of long dsRNA. The patent application publication alsoteaches that chemically synthesized oligonucleotides of the same orsimilar nature can also be used to target specific mRNAs for degradationin mammalian cells.

International Patent Application Publication No. WO 2002/44321 disclosesthat double-stranded RNA (dsRNA) 19-23 nt in length inducessequence-specific post-transcriptional gene silencing in a Drosophila invitro system. The PCT publication teaches that short interfering RNAs(siRNAs) generated by an RNase III-like processing reaction from longdsRNA or chemically synthesized siRNA duplexes with overhanging 3′ endsmediate efficient target RNA cleavage in the lysate, and the cleavagesite is located near the center of the region spanned by the guidingsiRNA.

U.S. Patent Application Publication No. US 2002/016216 discloses amethod for attenuating expression of a target gene in cultured cells byintroducing double stranded RNA (dsRNA) that comprises a nucleotidesequence that hybridizes under stringent conditions to a nucleotidesequence of the target gene into the cells in an amount sufficient toattenuate expression of the target gene.

International Patent Application Publication No. WO 2003/006477discloses engineered RNA precursors that when expressed in a cell areprocessed by the cell to produce targeted small interfering RNAs(siRNAs) that selectively silence targeted genes (by cleaving specificmRNAs) using the cell's own RNA interference (RNAi) pathway. The PCTpublication teaches that by introducing nucleic acid molecules thatencode these engineered RNA precursors into cells in vivo withappropriate regulatory sequences, expression of the engineered RNAprecursors can be selectively controlled both temporally and spatially,i.e., at particular times and/or in particular tissues, organs, orcells.

International Patent Application Publication No. WO 02/44321 disclosesthat double-stranded RNAs (dsRNAs) of 19-23 nt in length inducesequence-specific post-transcriptional gene silencing in a Drosophila invitro system. The PCT publication teaches that siRNAs duplexes can begenerated by an RNase III-like processing reaction from long dsRNAs orby chemically synthesized siRNA duplexes with overhanging 3′ endsmediating efficient target RNA cleavage in the lysate where the cleavagesite is located near the center of the region spanned by the guidingsiRNA. The PCT publication also provides evidence that the direction ofdsRNA processing determines whether sense or antisense-identical targetRNA can be cleaved by the produced siRNA complex. Systematic analyses ofthe effects of length, secondary structure, sugar backbone and sequencespecificity of siRNAs on RNA interference have been disclosed to aidsiRNA design. In addition, silencing efficacy has been shown tocorrelate with the GC content of the 5′ and 3′ regions of the 19 basepair target sequence. It was found that siRNAs targeting sequences witha GC rich 5′ and GC poor 3′ perform the best. More detailed discussionmay be found in Elbashir et al., 2001, EMBO J. 20:6877-6888 andAza-Blanc et al., 2003, Mol. Cell 12:627-637; each of which is herebyincorporated by reference herein in its entirety.

The invention provides specific siRNAs to target cellular components andinhibit virus replication as follows:

TABLE 2 Targets for Inhibition of Virus Replication and Inhibitory Polynucleotides SEQ SEQ Gene Symbol siRNA ID siRNA ID (Accession No.)(5′ to 3′ sense) NO (5′ to 3′ antisense) NO ACACA, GUUUGAUUGUGCCAUACUUTT1 AAGUAUGGCACAAUCAAACTT 2 transcript CAUGUCUGGCUUGCACCUATT 3UAGGUGCAAGCCAGACAUGTT 4 variant 6 GAUUGAGAAGGUUCUUAUUTT 5AAUAAGAACCUUCUCAAUCTT 6 (NM_000664) ACSL1 GUGUGAAGAAGAAAGCUCATT 7UGAGCUUUCUUCUUCACACTT 8 (NM_001995) GAACAAGGAUGCUUUGCUUTT 9AAGCAAAGCAUCCUUGUUCTT 10 GAAAUGAAGCCAUCACGUATT 11 UACGUGAUGGCUUCAUUUCTT12 AGPAT7 CCCUCUAUGCCAACAAUGUTT 13 ACAUUGUUGGCAUAGAGGGTT 14 (NM_153613)GGGUUUGGUGGACUUCCGATT 15 UCGGAAGUCCACCAAACCCTT 16 CCAACAAUGUUCAGAGGGUTT17 ACCCUCUGAACAUUGUUGGTT 18 AGXT2 CAAGCUAAAGAUCAGUAUATT 19UAUACUGAUCUUUAGCUUGTT 20 (NM_031900) GUGUGAAUGGAGUUGUCCATT 21UGGACAACUCCAUUCACACTT 22 GUCUCAUGAUAGGCAUAGATT 23 UCUAUGCCUAUCAUGAGACTT24 AGXT2L1 CACCUAUGUGCUUCACUGATT 25 UCAGUGAAGCACAUAGGUGTT 26 (NM_031279)GGAAUUGUCAGUUUAGAUUTT 27 AAUCUAAACUGACAAUUCCTT 28 GGUUAAUAGCUCUAUUAUATT29 UAUAAUAGAGCUAUUAACCTT 30 ART1 CAACUGCGAGUACAUCAAATT 31UUUGAUGUACUCGCAGUUGTT 32 (NM_004314) CCAACCAGGUGUAUGCAGATT 33UCUGCAUACACCUGGUUGGTT 34 CAAGUCUGGGCCUUGCCAUTT 35 AUGGCAAGGCCCAGACUUGTT36 ART3 GCCAUUAUGAGUGUGCAUUTT 37 AAUGCACACUCAUAAUGGCTT 38 (NM_001179)GCCAAAUGGGCAGCCCGAATT 39 UUCGGGCUGCCCAUUUGGCTT 40 CUCAAAUCUUUCUCCCUAUTT41 AUAGGGAGAAAGAUUUGAGTT 42 CARM1 GUAACCUCCUGGAUCUGAATT 43UUCAGAUCCAGGAGGUUACTT 44 (NM_199141) CCAGUAACCUCCUGGAUCUTT 45AGAUCCAGGAGGUUACUGGTT 46 CCUAUGACUUGAGCAGUGUTT 47 ACACUGCUCAAGUCAUAGGTT48 CDY2A GUAAUUAAAGAAAUGGUUATT 49 UAACCAUUUCUUUAAUUACTT 50 (NM_004825)GCUAUCAACUAGAUCGACATT 51 UGUCGAUCUAGUUGAUAGCTT 52 GAUAAUAAAUUCAACUAUUTT53 AAUAGUUGAAUUUAUUAUCTT 54 ELOVL2 GCUACAACUUACAGUGUCATT 55UGACACUGUAAGUUGUAGCTT 56 (NM_017770) CAAAGUUUCUUUGGACCAATT 57UUGGUCCAAAGAAACUUUGTT 58 CGUUAGUCAUCCUCUUCUUTT 59 AAGAAGAGGAUGACUAACGTT60 ELOVL3 GGAGUAUUGGGCAACCUCATT 61 UGAGGUUGCCCAAUACUCCTT 62 (NM_152310)GAAUGAUUAGGUUGCCUUATT 63 UAAGGCAACCUAAUCAUUCTT 64 CACUUAUUCUGGUCCUUCATT65 UGAAGGACCAGAAUAAGUGTT 66 ELOVL6 GGCUUAUGCAUUUGUGCUATT 67UAGCACAAAUGCAUAAGCCTT 68 (NM_024090) CAAUGGACCUGUCAGCAAATT 69UUUGCUGACAGGUCCAUUGTT 70 CAUGUCAGUGUUGACUUUATT 71 UAAAGUCAACACUGACAUGTT72 F13A1 CUAACAAGGUGGACCACCATT 73 UGGUGGUCCACCUUGUUAGTT 74 (NM_000129)CUAACCAUCCCUGAGAUCATT 75 UGAUCUCAGGGAUGGUUAGTT 76 GCCUAUAGUCUCAGAGUUATT77 UAACUCUGAGACUAUAGGCTT 78 GATM GAGACAUCCUGAUAGUUGUTT 79ACAACUAUCAGGAUGUCUCTT 80 (NM_001482) CAAAUGGCUUUCCAUGAAUTT 81AUUCAUGGAAAGCCAUUUGTT 82 CAUUAAAGUUAACAUUCGUTT 83 ACGAAUGUUAACUUUAAUGTT84 GGT3 CACUCAUGACUGAGGUCAUTT 85 AUGACCUCAGUCAUGAGUGTT 86 (NR_003267)CCUGUCUUGUGUGAGGUGUTT 87 ACACCUCACACAAGACAGGTT 88 CCAGCAUUCACCAAUGAGUTT89 ACUCAUUGGUGAAUGCUGGTT 90 GPAM GUUAUUAGAAUGUUACGAATT 91UUCGUAACAUUCUAAUAACTT 92 (NM_020918) GAGUGUAGCAAGAGGUGUUTT 93AACACCUCUUGCUACACUCTT 94 GCAUGUUUGCCACCAAUGUTT 95 ACAUUGGUGGCAAACAUGCTT96 HS6ST1 GACGUCUUUGCAUAUGUGUTT 97 ACACAUAUGCAAAGACGUCTT 98 (NM_004807)CUGUUCGAGCGGACGUUCATT 99 UGAACGUCCGCUCGAACAGTT 100 CAGUACCUGUUCGAGCGGATT101 UCCGCUCGAACAGGUACUGTT 102 HS6ST2 GCCAUUUACCCAGUAUAAUTT 103AUUAUACUGGGUAAAUGGCTT 104 (NM_147175) GGUAUCAGUUUAUGAGGCATT 105UGCCUCAUAAACUGAUACCTT 106 CAUGAACUUUAUUUCGCCATT 107UGGCGAAAUAAAGUUCAUGTT 108 L00541473 GUCCCUGUACGAGCGGUUATT 109UAACCGCUCGUACAGGGACTT 110 (NR_003602) GCGGUUAAGUCAGAGGAUGTT 111CAUCCUCUGACUUAACCGCTT 112 CUGUACGAGCGGUUAAGUCTT 113GACUUAACCGCUCGUACAGTT 114 LTC4S GCGAGUACUUCCCGCUGUUTT 115AACAGCGGGAAGUACUCGCTT 116 (NM_000897) GCCGGCAUCUUCUUUCAUGTT 117CAUGAAAGAAGAUGCCGGCTT 118 GGGUCGCCGGCAUCUUCUUTT 119AAGAAGAUGCCGGCGACCCTT 120 MCCC2 CCAAGAUUUCUCUACAUUUTT 121AAAUGUAGAGAAAUCUUGGTT 122 (NM_022132) GAUUUAUGGUUGGUAGAGATT 123UCUCUACCAACCAUAAAUCTT 124 CAUCAUGCCCUUCACUUAATT 125UUAAGUGAAGGGCAUGAUGTT 126 MGST3 GUGUAUCCUCCCUUCUUAUTT 127AUAAGAAGGGAGGAUACACTT 128 (NM_004528) CUGGAUUGUUGGACGAGUUTT 129AACUCGUCCAACAAUCCAGTT 130 GUGUUUACCACCCGCGUAUTT 131AUACGCGGGUGGUAAACACTT 132 PDIA6 CAUCGAAUUUCAACCGAGATT 133UCUCGGUUGAAAUUCGAUGTT 134 (NM_005742) GUGAUAGUUCAAGUAAGAATT 135UUCUUACUUGAACUAUCACTT 136 CCAUCAAUGCACGCAAGAUTT 137AUCUUGCGUGCAUUGAUGGTT 138 PLA2G7 CAGAGAUUCAGAUGUGGUATT 139UACCACAUCUGAAUCUCUGTT 140 (NM_005084) GCCUUAUUCCGUUGGUUGUTT 141ACAACCAACGGAAUAAGGCTT 142 GAAAUGAGCAGGUACGGCATT 143UGCCGUACCUGCUCAUUUCTT 144 PNMT CCUUCAACUGGAGCAUGUATT 145UACAUGCUCCAGUUGAAGGTT 146 (NM_002686) GACAUCACCAUGACAGAUUTT 147AAUCUGUCAUGGUGAUGUCTT 148 CCCUCAUCGACAUUGGUUCTT 149GAACCAAUGUCGAUGAGGGTT 150 SLC27A3 GCAACGUGGCCACCAUCAATT 151UUGAUGGUGGCCACGUUGCTT 152 (NM_024330) CCAGAUACCUGGGAGCGUUTT 153AACGCUCCCAGGUAUCUGGTT 154 CGCUGAAGUGGAUGGGCCATT 155UGGCCCAUCCACUUCAGCGTT 156 TALDO1 CACAAGAGGACCAGAUUAATT 157UUAAUCUGGUCCUCUUGUGTT 158 (NM_006755) GCAACACGGGCGAGAUCAATT 159UUGAUCUCGCCCGUGUUGCTT 160 CGAAUUCUUAUAAAGCUGUTT 161ACAGCUUUAUAAGAAUUCGTT 162 TKTL1 GUCGUUUGUGGAUGUGGCATT 163UGCCACAUCCACAAACGACTT 164 CAUGCAAAGCCAAUGCCGATT 165UCGGCAUUGGCUUUGCAUGTT 166 GGUAUUCUGGCAGGCUUCUTT 167AGAAGCCUGCCAGAAUACCTT 168 UGT3A2 GUUUCUAUUCAGUUAAAGATT 169UCUUUAACUGAAUAGAAACTT 170 (NM_174914) GAGACAUUGGCUCUUAAGATT 171UCUUAAGAGCCAAUGUCUCTT 172 GAACUUCGACAUGGUGAUATT 173UAUCACCAUGUCGAAGUUCTT 174 UST CCUAUUUAUUCACUCGACATT 175UGUCGAGUGAAUAAAUAGGTT 176 (NM_005715) GAGAUACGAGUACGAGUUUTT 177AAACUCGUACUCGUAUCUCTT 178 CCUUAAGGGACUAAAUUAATT 179UUAAUUUAGUCCCUUAAGGTT 180 SOAT1 CGUCAUACUCCAACUAUUATT 196UAAUAGUUGGAGUAUGACGTT 197 (NM_003101) CAAAUCUGCUGCCAUGUUATT 198UAACAUGGCAGCAGAUUUGTT 199 CGAAUAUGCCUUGGCUGUUTT 200AACAGCCAAGGCAUAUUCGTT 201 SOAT2 GCUAUACAAUCCUACCCAU 202AUGGGUAGGAUUGUAUAGC 203 (NM_003101) CUGAUACUCUUCCUUGUCA 204UGACAAGGAAGAGUAUCAG 205 CGAUCUUGGUCCUGCCAUA 206 UAUGGCAGGACCAAGAUCG 207CA7 CCAGUUUGCUCCUUGGUCATT 208 UGACCAAGGAGCAAACUGGTT 209 (NM_005182)CACUGAAGGGCCGCGUGGUTT 210 ACCACGCGGCCCUUCAGUGTT 211GAGACUCAAGCAAUAAUUATT 212 UAAUUAUUGCUUGAGUCUCTT 213 OTOP3CCCUGAAUGUGGUGUUCCUTT 214 AGGAACACCACAUUCAGGGTT 215 (NM_178233)GAGGCUUCCUGAUGCUCUATT 216 UAGAGCAUCAGGAAGCCUCTT 217GGCAAUGAGACCAACACCUTT 218 AGGUGUUGGUCUCAUUGCCTT 219 TBXAS1CAAUAAGAACCGAGACGAATT 220 UUCGUCUCGGUUCUUAUUGTT 221 (NM_001061)GUGAAACACUGCAAGCGUUTT 222 AACGCUUGCAGUGUUUCACTT 223GAGACUUCCUCCAAAUGGUTT 224 ACCAUUUGGAGGAAGUCUCTT 225 TYMSCAAUGGAUCCCGAGACUUUTT 226 AAAGUCUCGGGAUCCAUUGTT 227 (NM_001071)GUACAAUCCGCAUCCAACUTT 228 AGUUGGAUGCGGAUUGUACTT 229GAGAUAUGGAAUCAGAUUATT 230 UAAUCUGAUUCCAUAUCUCTT 231 TXNDC11GCAUAGAAUGCAGCAAUUUTT 232 AAAUUGCUGCAUUCUAUGCTT 233 (NM_01015914)GAAAGAAUUUGCGGCAAUUTT 234 AAUUGCCGCAAAUUCUUUCTT 235CAGAGUACGUUCGACGGGATT 236 UCCCGUCGAACGUACUCUGTT 237 PDIA5GACGGUUCUUGUUCCAGUATT 238 UACUGGAACAAGAACCGUCTT 239 (NM_006810)CCAUUACCAGGAUGGUGCATT 2406 UGCACCAUCCUGGUAAUGGTT 241CCGUUUAUCACCUGACCGATT 242 UCGGUCAGGUGAUAAACGGTT 243 PTGS2GAGUAUGCGAUGUGCUUAATT 244 UUAAGCACAUCGCAUACUCTT 245 (NM_000963)CAGUAUAAGUGCGAUUGUATT 246 UACAAUCGCACUUAUACUGTT 247GUAUGAGUGUGGGAUUUGATT 248 UCAAAUCCCACACUCAUACTT 249 STX8GACUACUUCUGGCAUCCUUTT 250 AAGGAUGCCAGAAGUAGUCTT 251 (NM_004853)CAACCUAGUGGAGAACACATT 252 UGUGUUCUCCACUAGGUUGTT 253CAAAGCUUACCGUGACAAUTT 254 AUUGUCACGGUAAGCUUUGTT 255 OTOP2CUGUCAGCCUCUUCCGGGATT 256 UCCCGGAAGAGGCUGACAGTT 257 (NM_178160)CCCUUCAGACCAGCGGGAATT 258 UUCCCGCUGGUCUGAAGGGTT 259CUGACCUGGUGUGGUCUCATT 260 UGAGACCACACCAGGUCAGTT 261 STX6CAAGUUGUCAGGGACAUGATT 262 UCAUGUCCCUGACAACUUGTT 263 (NM_005819)GAAAUAACCUCCGGAGCAUTT 264 AUGCUCCGGAGGUUAUUUCTT 265CAGUUAUGUUGGAAGAUUUTT 266 AAAUCUUCCAACAUAACUGTT 267

In addition, siRNA design algorithms are disclosed in PCT publicationsWO 2005/018534 A2 and WO 2005/042708 A2; each of which is herebyincorporated by reference herein in its entirety. Specifically,International Patent Application Publication No. WO 2005/018534 A2discloses methods and compositions for gene silencing using siRNA havingpartial sequence homology to its target gene. The application providesmethods for identifying common and/or differential responses todifferent siRNAs targeting a gene. The application also provides methodsfor evaluating the relative activity of the two strands of an siRNA. Theapplication further provides methods of using siRNAs as therapeutics fortreatment of diseases. International Patent Application Publication No.WO 2005/042708 A2 provides a method for identifying siRNA target motifsin a transcript using a position-specific score matrix approach. It alsoprovides a method for identifying off-target genes of an siRNA using aposition-specific score matrix approach. The application furtherprovides a method for designing siRNAs with improved silencing efficacyand specificity as well as a library of exemplary siRNAs.

Design software can be use to identify potential sequences within thetarget enzyme mRNA that can be targeted with siRNAs in the methodsdescribed herein. See, for example,http://www.ambion.com/techlib/misc/siRNA_finder.html (“Ambion siRNATarget Finder Software”). For example, the nucleotide sequence of ACSL1,which is known in the art (GenBank Accession No. NM_001995) is enteredinto the Ambion siRNA Target Finder Software(http://www.ambion.com/techlib/misc/siRNA_finder.html), and the softwareidentifies potential ACSL1 target sequences and corresponding siRNAsequences that can be used in assays to inhibit human ACSL1 activity bydownregulation of ACSL1 expression. Using this method, non-limitingexamples of ACSL1 target sequence (5′ to 3′) and corresponding sense andantisense strand siRNA sequences (5′ to 3′) for inhibiting ACSL1 areidentified and presented below:

ACSL1 Target  Sense Strand  Antisense  Sequence siRNA Strand siRNA 1.AAGAACCAAGGGC GAACCAAGGGCAUAUA CUUUAUAUGCCCUUGG ATATAAAG AAGtt UUCtt(SEQ ID NO:  (SEQ ID NO: 182) (SEQ ID NO: 183) 181) 2. AACCAAGGGCATACCAAGGGCAUAUAAAG UGUCUUUAUAUGCCCU TAAAGACA ACAtt UGGtt (SEQ ID NO: (SEQ ID NO: 185) (SEQ ID NO: 186) 184) 3. AAGGGCATATAAA GGGCAUAUAAAGACAGCAUCUGUCUUUAUAUG GACAGATG AUGtt CCCtt (SEQ ID NO:  (SEQ ID NO: 188)(SEQ ID NO: 189) 187) 4. AAAGACAGATGGG AGACAGAUGGGAGGAG GGUCUCCUCCCAUCUGAGGAGACC ACCtt UCUtt (SEQ ID NO:  (SEQ ID NO: 191) (SEQ ID NO: 192) 190)5. AAGAAGCATCTAC GAAGCAUCUACAUAGG GUACCUAUGUAGAUGC ATAGGTAC UACtt UUCtt(SEQ ID NO:  (SEQ ID NO: 194) (SEQ ID NO: 195) 193)

The same method can be applied to identify target sequences of anyenzyme and the corresponding siRNA sequences (sense and antisensestrands) to obtain RNAi molecules.

In certain embodiments, a compound is an siRNA effective to inhibitexpression of a target enzyme, e.g., ACSL1 or ART1, wherein the siRNAcomprises a first strand comprising a sense sequence of the targetenzyme mRNA and a second strand comprising a complement of the sensesequence of the target enzyme, and wherein the first and second strandsare about 21 to 23 nucleotides in length. In some embodiments, the siRNAcomprises first and second strands comprise sense and complementsequences, respectively, of the target enzyme mRNA that is about 17, 18,19, or 20 nucleotides in length.

The RNAi molecule (e.g., siRNA, shRNA, miRNA) can be both partially orcompletely double-stranded, and can encompass fragments of at least 18,at least 19, at least 20, at least 21, at least 22, at least 23, atleast 24, at least 25, at least 30, at least 35, at least 40, at least45, and at least 50 or more nucleotides per strand. The RNAi molecule(e.g., siRNA, shRNA, miRNA) can also comprise 3′ overhangs of at least1, at least 2, at least 3, or at least 4 nucleotides. The RNAi molecule(e.g., siRNA, shRNA, miRNA) can be of any length desired by the user aslong as the ability to inhibit target gene expression is preserved.

RNAi molecules can be obtained using any of a number of techniques knownto those of ordinary skill in the art. Generally, production of RNAimolecules can be carried out by chemical synthetic methods or byrecombinant nucleic acid techniques. Methods of preparing a dsRNA aredescribed, for example, in Ausubel et al., Current Protocols inMolecular Biology (Supplement 56), John Wiley & Sons, New York (2001);Sambrook et al., Molecular Cloning: A Laboratory Manual, 3.sup.rd ed.,Cold Spring Harbor Press, Cold Spring Harbor (2001); and can be employedin the methods described herein. For example, RNA can be transcribedfrom PCR products, followed by gel purification. Standard proceduresknown in the art for in vitro transcription of RNA from PCR templates.For example, dsRNA can be synthesized using a PCR template and theAmbion T7 MEGASCRIPT, or other similar, kit (Austin, Tex.); the RNA canbe subsequently precipitated with LiC1 and resuspended in a buffersolution.

To assay for RNAi activity in cells, any of a number of techniques knownto those of ordinary skill in the art can be employed. For example, theRNAi molecules are introduced into cells, and the expression level ofthe target enzyme can be assayed using assays known in the art, e.g.,ELISA and immunoblotting. Also, the mRNA transcript level of the targetenzyme can be assayed using methods known in the art, e.g., Northernblot assays and quantitative real-time PCR. Further the activity of thetarget enzyme can be assayed using methods known in the art and/ordescribed herein in section 5.3. In a specific embodiment, the RNAimolecule reduces the protein expression level of the target enzyme by atleast about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. In oneembodiment, the RNAi molecule reduces the mRNA transcript level of thetarget enzyme by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or 95%. In a particular embodiment, the RNAi molecule reduces theenzymatic activity of the target enzyme by at least about 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, or 95%.

2.1.2 Small Molecules

2.1.2.1 Triacsin Compounds

In one embodiment, the present invention provides a method of treatingor preventing a viral infection in a mammal, comprising administering toa subject in need therefore a therapeutically effective amount oftriacsin C or a relative, analogue, or derivative thereof

Triacsin C exists in two tautomeric forms as follows:

Triacsin C is a fungal antimetabolite that inhibits long chain acyl-CoAsynthetases (ACSLs), arachidonoyl-CoA synthetase, and triglyceride andcholesterol ester biosynthesis. It is a member of a family of relatedcompounds (Triacsins A-D) isolated from the culture filtrate ofStreptomyces sp. SK-1894 (Omura et al., J Antibiot 39, 1211-8, 1986;Tomoda et al., Biochim Biophys Acta, 921, 595-8, 1987), all of whichconsist of 11-carbon alkenyl chains with a common triazenol moiety attheir termini. Structures of of triacsins A, B, and D are as follows:

According to the invention, Triacsin C or a related compound or analogor prodrug thereof, is used for treating or preventing infection by awide range of viruses, such as, but not limited to, DNA viruses (doublestranded and single stranded), double-stranded RNA viruses,single-stranded RNA viruses (negative-sense and positive-sense),single-stranded RNA retroviruses, and double stranded viruses with RNAintermediates. For example, as exemplified herein, nanomolarconcentrations of triacsin C inhibit the replication of HCMV (aHerpesvirus; comprising a double stranded DNA genome), herpes simplexvirus-1 (HSV-1), influenza A (an Orthomyxovirus; a negative-sensesingle-stranded RNA virus) and hepatitis C virus (HCV). Further,triacsin C exhibits broad spectrum anti-viral activity against envelopedviruses. Accordingly, in one embodiment of the invention, Triacsin C isused for treating or preventing infection by an enveloped virus. Also,triacsin C is active against non-enveloped viruses whose replicationoccurs on host cell membrane structures and against viruses that induceincreases in host cell membrane.

Triacsin C inhibits ACSLs and also inhibits arachidonoyl-CoA synthase.Triacsin C inhibits triacylglycerol (TG) and cholesterol ester (CE)synthesis with an IC₅₀ of 100 nM and 190 nM, respectively. Triacsin Cinhibits ACSLs in rat liver cell sonicates with an IC₅₀ of about 8.7 μMand also inhibits arachidonoyl-CoA sythethase.

Nanomolar concentrations of triacsin C inhibited by >10-fold thereplication of 3 of 4 viruses tested: HCMV, herpes simplex virus-1(HSV-1), and influenza A (but not adenovirus). HCMV, HSV-1, andinfluenza A (but not adenovirus) have a lipid envelope. See Example 1.

Triacsin C relatives that the present invention include withoutlimitation triacsins A, C, D and WS-1228 A and B (Omura et al., JAntibiot 39, 1211-8, 1986). Triacsin C analogues of the presentinvention include without limitation 3 to 25 carbon unbranched (linear)carbon chains with the triazenol moiety of triacsin C at their terminiand with any combination of cis or trans double bonds in the carbonchain. In certain embodiments of the invention, the carbon chain is noshorter than 4, 5, 6, 7, 8, 9, 10, or 11 carbon atoms. In certainembodiments, the carbon chain is no longer than 24, 23, 22, 21, 20, 19,18, 17, 16, 15, 14, 13, 12, or 11 atoms. In certain embodiments, thecarbon chain contains exactly 0, 1, 2, 3, or 4 cis double bonds. Incertain embodiments, the carbon chain contains exactly 0, 1, 2, 3, 4, 5,or 6 trans double bonds. In certain embodiments, as in triacsin C, thereis a trans double bond at the 2^(nd) carbon-carbon bond in the chain(numbering where the carbon-nitrogen bound is bond 0). In otherembodiments, there are one or more trans double bonds at bonds 3, 4, 5,6, 7, 8, 9, 10, 11, or 12 in the chain. In certain embodiments, as intriacsin C, there is a cis-double bond at the 7^(th) carbon-carbon bondin the chain. In other embodiments, there are one or more cis doublebonds at bonds 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 in the chain. TriacsinC derivatives of the present invention include without limitationtriacsin or its analogues with insertion of heteroatoms or methyl orethyl groups in place of hydrogen atoms at any point in the carbonchain. They further include variants where a portion of the linear chainof carbon-carbon bonds is replaced by one or more 3, 4, 5, or 6 memberedrings, comprised of saturated or unsaturated carbon atoms orheteroatoms. A synthetic route to this class of compounds is describedin U.S. Pat. No. 4,297,096 to Yoshida et al.

In certain embodiments, the triacin analogs of the invention includecompounds of formula I:

wherein R¹ is a carbon chain having from 3 to 23 atoms (includingoptional heteroatoms) in the chain, wherein the chain comprises0-10 double bonds within the chain; and0-4 heteroatoms within the chain;and wherein 0-8 of the carbon atoms of R¹ are optionally substituted.

If one or more optional heteroatoms occur within the R¹ chain, inpreferred embodiments each heteroatom is independently selected from O,S, and NR², wherein R² is selected from H, C₁₋₆ alkyl, and C₃₋₆cycloalkyl.

When the carbon atoms of R¹ are substituted, it is preferred that from0-8 hydrogen atoms along the chain may be replaced by a substituentselected from halo, OR², SR², lower alkyl, and cycloalkyl, wherein R² isH, C₁₋₆ alkyl, and C₃₋₆ cycloalkyl. In certain preferred embodiments, R¹is unsubstituted (i.e., R¹ is unbranched, and none of the hydrogens havebeen replaced by a substituent).

In preferred embodiments for compounds of the formula I, R¹ has a chainlength of 8 to 12 atoms. More preferably, R¹ has a total chain length ofR¹ has a chain length of 9 to 11 atoms. Most preferably R¹ has a chainlength of 10 atoms. In other preferred embodiments, R¹ has 2 to 4 doublebonds.

In certain embodiments, the triacin anolog is selected from

In certain embodiments, the triacin analogs of the invention includecompounds of formula II:

wherein R is selected from C₁₋₆ alkyl; andwherein R₆ and R_(6′) are independently selected from H, C₁₋₃ alkyl; orR₆ and R_(6′) taken together form a cycloalkyl group of formula—(CH₂)_(n) wherein n is 2-6. In certain embodiments R may be selectedfrom Me, Et, n-butyl, i-propyl, n-pentyl to n-hexyl. In certainembodiments, R₆ and R_(6′) are independently selected from Me and F; orR₆ and R_(6′) taken together form a cycloalkyl group of formula—(CH₂)_(n) wherein n is 2, 3, 4, and 6.

For example, in certain embodiments the triacin analog of formula II isone of the following compounds:

In certain embodiments, the triacin analogs of the invention includecompounds of formula III:

Wherein the Linker is selected from Z or E-olefin, alkyne, optionallysubstituted phenyl ring or optionally substituted heteroaryl ring (suchas pyridine).

For example, compounds of formula III include:

In another embodiment triacin analogs of the invention include compoundsof formula IVa and IVb:

Wherein R′ is C₁₋₄ alkyl. In certain embodiments R′ is Me, Et, nPr, iPr,nBu. In certain embodiments one of the phenyl carbons at positions 2-6may be replaced by N.

For example, in certain embodiments compounds of formula IVa include:

In certain embodiments compounds of formula IVb include:

In one embodiment triacsin C analogs are designed from correspondinglipophillic tail groups, spacer groups, and polar groups

wherein the lipophilic tail group is selected from the tail group oftraicin A-D and

wherein the spacer group is selected from the spacer group of traicinA-D and

andwherein the polar group is selected from the polar group of traicin A-Dand

In one embodiment, the triacin C analog composed of the tail, spacer andpolar group is

2.1.2.2 Inhibitors of Lipid Drop Formation

Inhibitors of lipid drop formation include, but are not limited to thefollowing compounds:

PF-1052 (CAS: 147317-15-5)

Spylidone (Liquid Droplet inhibition IC₅₀ 42 uM) (Tomoda et al., 2007,Pharmacol. Ther. 115:375-89);

Sespendole (Liquid Droplet inhibition IC50 4 uM) (Tomoda et al., 2007,Pharmacol. Ther. 115:375-89)

Terpendole C (Liquid Droplet inhibition IC₅₀ 2.5 μM) (Tomoda et al.,2007, Pharmacol. Ther. 115:375-89);

Compound 7 (Sastry et al., 2010, J. Org. Chem. 75:2274-80);

Rubimaillin;

Compound 8 (Ho, L. K. et al., 1996, J. Nat. Prod. 59:330-3); and

Compound 9 (Ho, L. K. et al., 1996, J. Nat. Prod. 59:330-3).

Analogs of PF-1052 and Spylidone useful in the present invention include

Additional inhibitors of lipid droplet formation include Vermisporin;Beauveriolides; Phenochalasins; Isobisvertinol; and K97-0239.

2.1.2.3 ACAT Inhibitors

In certain embodiments, the ACAT inhibitors of the invention includecompounds of formula V

whereinX and Y are independently selected from N and CH;R_(1′) and R_(2′) are independently selected from H, C₁₋₆ alkyl whichmay be optionally substituted with F, OCH₃ and OH, and C₁₋₆ cycloalkyl;R₆ and R₇ are independently selected from H, and C₁₋₃ alkyl, or R₆ andR₇ taken together may form a C₃₋₆ cycloalkyl,R₃, R₄ and R₅ are independently selected from H, C₁₋₆ alkyl which may beoptionally substituted with F, OCH₃ and OH, and C₁₋₆ cycloalkyl;additionally or alternatively, one of R₆ or R₇ may be taken togetherwith R₅ to form a C₅₋₁₁ cycloalkyl ring.In certain embodiments, R_(1′) and/or R_(2′) are independently selectedfrom branched C₃₋₅ alkyl and particularly isopropyl.In certain embodiments, R₃, R₄ and/or R₅ are independently selected frombranched C₃₋₅ alkyl and particularly isopropyl.

In certain embodiments, R₆ and R₇ are both H.

In certain embodiments, the ACAT inhibitors of the invention includecompounds of formula Va

whereinR_(1′) and R_(2′) are independently selected from H, C₁₋₆ alkyl whichmay be optionally substituted with F, OCH₃ and OH, and C₁₋₆ cycloalkyl;R₃ and R₄ are independently selected from H, C₁₋₆ alkyl which may beoptionally substituted with F, OCH₃ and OH, and C₁₋₆ cycloalkyl;n is selected from 1 to 7; andR₈ is selected from H and C₁₋₃ alkyl.In certain embodiments, R_(1′) and/or R_(2′) are independently selectedfrom branched C₃₋₅ alkyl and particularly isopropyl.In certain embodiments, R₃ and/or R₄ are independently selected frombranched C₃₋₅ alkyl and particularly isopropyl.In certain embodiments, R₈ is methyl.

In one embodiment the compound of formula V is

Avasimibe (ACAT IC₅₀ 479 nM).

Additional ACAT inhibitors of the invention include, but are not limitedto the following compounds:

Pactimibe (Liver ACAT IC₅₀ 312 nM) (Ohta et al., 2010, Chem. Pharm.Bull. 58:1066-76);

Compound 1 (Liver ACAT IC₅₀ 120 nM) (Takahashi et al., 2008, J. Med.Chem. 51:4823-33);

Compound 21 (Liver ACAT IC₅₀ 113 nM) (Ohta et al., 2010, Chem. Pharm.Bull. 58:1066-76);

Compound 12g (ACAT IC₅₀ 68 nM) (Asano et al., 2009, Bioorg. Med. Chem.Lett. 19:1062-5);

SMP-797 (ACAT IC₅₀ 31 nM) (Asano et al., 2009, Bioorg. Med. Chem. Lett.19:1062-5);

CL-283,546 (Liquid Droplet inhibition IC₅₀ 35 nM) (Tomoda et al., 2007,Pharmacol. Ther. 115:375-89);

Wu-V-23 (Tomoda et al., 2007, Pharmacol. Ther. 115:375-89); and

Eflucimibe.

2.1.2.4 Elongase Inhibitors

One example of an elongase inhibitor is a compound of formula VI:

wherein L is selected from carbamate, urea, or amide including, forexample

and wherein R is selected from halo; CF₃; cyclopropyl; optionallysubstituted C₁₋₅ alkyl, wherein the C₁₋₅ alkyl may be substituted withhalo, oxo, —OH, —CN, —NH₂, CO₂H, and C₁₋₃ alkoxy;wherein R₁ is selected from substituted phenyl where the substituentsare selected from F, CF₃, Me, OMe, or isopropyl;wherein R₂ is Cl, Ph, 1-(2-pyridone), 4-isoxazol, 3-pyrazol, 4-pyrazol,1-pyrazol, 5-(1,2,4-triazol), 1-(1,2,4-triaol), 2-imidazolo,1-(2-pyrrolidone), 3-(1,3-oxazolidin-2-one).The chiral center at C4 can be racemic, (S), (R), or any ratio ofenantiomers. In one embodiment, L is an amide. In certain embodiments, Ris selected from Cl, CF₃, methyl, ethyl, isopropyl and, cyclopropyl. Incertain embodiments R1 is para-substituted wherein the substituent isselected from F, CF₃, Me, OMe, or isopropyl,

In one embodiment, the compound of formula VIa is

wherein R is selected from

In another embodiment, the elongase inhibitor is a compound of formulaVIb

wherein R¹ is substituted at position 2, 3, or 4 with F, or Me, or R¹ issubstituted at position 4 with MeO, or CF₃. R² is Cl, H, Ph, 4-isoxazol,4-pyrazol, 3-pyrazol, 1-pyrazol, 5-(1,2,4-triazol), 1-(1,2,4-triazol),2-imidazol, 1-(2-pyrrolidone), or 3-(1,3-oxazolidin-2-one). In oneembodiment the compound of formula VI is

(S)-y. (See, Mizutani et al., 2009, J. Med. Chem. 52:7289-7300).

In another embodiment, the compound of formula VI is

Additional examples of an elongase inhibitors are compounds of formulaVIIa and VIIb

wherein R₁ is selected from OMe, OiPr, OCF₃, OPh, CH₂Ph, F, CH₃, CF₃,and benzyl; and

wherein R₂ is selected from C₁₋₄ alkyl (such as nBu, nPr, and iPr);phenyl; substituted phenyl where substitutents are selected from OMe,CF₃, F, tBu, iPr and thio; 2-pyridine; 3-pyridine; and N-methyimidazole. (See, Sasaki et al., 2009, Biorg. Med. Chem. 17:5639-47).

In one embodiment, R₁ is selected from OiPr and OCF₃. In one embodimentR₂ is selected from nBu, unsubstituted phenyl, fluorophenyl andthiophenyl.

In one embodiment the inhibitor of formula VIIa is

wherein R² is selected from butyl, propyl, phenyl, pyridyl, andimidazole.

In one embodiment the inhibitor of formula VIIa is selected from

which has hELOVL6 IC₅₀ of 1710 nM;

which has hELOVL6 IC₅₀ of 220 nM and a hELOVL3 IC₅₀ of 1510 nM; and

which has hELOVL6 IC₅₀ of 930 nM.

Yet another example of an elongase inhibitor is a compound of formulaVIII

wherein R₁ is selected from H, unsubtitued phenyl; substituted phenylwhere substitutents are selected from F, Me, Et, Cl, OMe, OCF₃, and CF₃;C₁₋₆ alkyl (such as Me, Et, iPr, and n-propyl); and C₃₋₆ cycloalkyl(cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl);wherein R₃ and R₄ are independently selected from H; C₁₋₃ alkyl; andphenyl; or R₃ and R₄ taken together form a cycloalkyl of formula—(CH2)_(n)— where n=2, 3, 4 and 5;wherein R₅ is selected from methyl; CF₃; cyclopropyl; unsubtituedphenyl; mono- and disubstituted phenyl where substitutents are selectedfrom F, Me, Et, CN, iPr, Cl, OMe, OPh, OCF₃, and CF₃; unsubstitutedheteroaromatic groups (such as 2, 3, or 4-pyridine, isoxazol, pyrazol,triazol); and imidazolo.

In other embodiments the compound of formula VIII is

wherein R⁵ is a substituted phenyl ring, including, but not limited to

(See Takahashi et al., 2009, J. Med. Chem. 52:3142-5.)

In other embodiments compound of formula VIII is one of the followingcompounds:

which has a hELOVL6 IC50 of 290 nM,

which has a hELOVL6 IC50 of 10 nM and a hELOVL3 IC50 of 59 nM, and

Compound 37, which has a hELOVL6 IC50 of 8.9 nM and a hELOVL3 IC50 of337 nM.

In one embodiment the elongase inhibitor is a compound of formula IX

wherein L is selected from urea or an amide, for example

wherein R₁ is selected form 2-, 3-, and 4-pyridine; pyrimidine;unsubstituted heteroaryls such as isoxazol, pyrazol, triazol, imidazole;and unsubstituted phenyl; ortho, meta or para-substituted phenyl wheresubstitutents are F, Me, Et, Cl, OMe, OCF₃, and CF₃, Cl, iPr and phenyl;wherein R₂ is selected from Cl; iPr; phenyl; ortho, meta orpara-substituted phenyl where substitutents are F, Me, Et, Cl, OMe,OCF₃, and CF₃; and heteroaryls such as 2-, 3-, and 4-pyridine,pyrimidine, and isoxazol, pyrazol, triazol, and imidazo.

In one embodiment L is urea. In one embodiment, R₁ is para-substitutedCF₃ phenyl. In one embodiment, R₂ is phenyl. In another embodiment, R₂is 2-pyridyl.

In one embodiment the compound of formula IX is selected from

(endo-1w) which has a hELOVL6 IC50 of 79 nM and a hELOVL3 IC50 of 6940nM, and

(endo-1k) which has a hELOVL6 IC50 of 78 nM.

2.1.2.5 ART1 Inhibitors

Meta-iodo-benzylguanidine (MIBG) is an inhibitor ofADP-ribosyltransferase 1 (ART1). 50 μM MIBG reduced HCMV titer frominfected MRC5 fibroblasts by about 70% with little or no effect on cellmorphology.

2.1.2.6 AGXT2 Inhibitors

Aminooxyacetic acid (AOAA) is an inhibitor of alanine-glyoxylateaminotransferase 2 (AGXT2). 0.5 mM AOAA decreases HCMV replication by100-fold with no measurable decrease in cell viability at concentrationsup to 2.5 mM. 0.5 mM and 1 mM AOAA decreases influenza A replication inMDCK cells by at least 1000-fold after 24 hours with no evidence of hostcell toxicity. 0.5 mM and 1 mM concentrations of AOAA decreaseadenovirus titer in MRC2 cells by 20-fold and 500-fold respectively.

3. Screening Assays to Identify Inhibitors of Host Cell Target Enzymes

Compounds known to be inhibitors of the host cell target enzymes can bedirectly screened for antiviral activity using assays known in the artand/or described infra (see, e.g., Section 4 et seq.). While optional,derivatives or congeners of such enzyme inhibitors, or any othercompound can be tested for their ability to modulate the enzyme targetsusing assays known to those of ordinary skill in the art and/ordescribed below. Compounds found to modulate these targets can befurther tested for antiviral activity. Compounds found to modulate thesetargets or to have antiviral activity (or both) can also be tested inthe metabolic flux assays described in Section 4.2.8 in order to confirmthe compound's effect on the metabolic flux of the cell. This isparticularly useful for determining the effect of the compound inblocking the ability of the virus to alter cellular metabolic flux, andto identify other possible metabolic pathways that may be targeted bythe compound.

Alternatively, compounds can be tested directly for antiviral activity.Those compounds which demonstrate anti-viral activity, or that are knownto be antiviral but have unacceptable specificity or toxicity, can bescreened against the enzyme targets of the invention. Antiviralcompounds that modulate the enzyme targets can be optimized for betteractivity profiles.

Any host cell enzyme, known in the art and/or described in Section 5.1,is contemplated as a potential target for antiviral intervention.Further, additional host cell enzymes that have a role, directly orindirectly, in regulating the cell's metabolism are contemplated aspotential targets for antiviral intervention. Compounds, such as thecompounds disclosed herein or any other compounds, e.g., a publiclyavailable library of compounds, can be tested for their ability tomodulate (activate or inhibit) the activity of these host cell enzymes.If a compound is found to modulate the activity of a particular enzyme,then a potential antiviral compound has been identified.

In one embodiment, an enzyme that affects or is involved in synthesis oflong and very long chain fatty acids is tested as a target for thecompound, for example, ACSL1, ELOVL2, ELOVL3, ELOVL6, or SLC27A3. In oneembodiment, long and very long chain acyl-CoA synthases are tested formodulation by the compound. In another embodiment, fatty acid elongasesare tested for modulation by the compound. In one embodiment, an enzymeinvolved in synthesis of cysteinyl leukotrienes is tested for modulationby the compound. In one embodiment, an enzyme that plays role in lipidstorage (including but not limited to ADP-ribosyltransferase 1 or 3) istested for modulation by the compound. In another embodiment, analanine-glyoxylate aminotransferase is tested for modulation by thecompound. In yet another embodiment, an enzyme in the pentosephosposphate pathway is is tested for modulation by the compound.

In preferred embodiments, a compound is tested for its ability tomodulate host metabolic enzymes by contacting a composition comprisingthe compound with a composition comprising the enzyme and measuring theenzyme's activity. If the enzyme's activity is altered in the presenceof the compound compared to a control, then the compound modulates theenzyme's activity. In some embodiments of the invention, the compoundincreases an enzyme's activity (for example, an enzyme that is anegative regulator of fatty acid biosynthesis might have its activityincreased by a potential antiviral compound). In specific embodiments,the compound increases an enzyme's activity by at least approximately10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% or 90%. In someembodiments, the compound decreases an enzyme's activity. In particularembodiments, the compound decreases an enzyme's activity by at leastapproximately 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%or 100%. In certain embodiments, the compound exclusively modulates asingle enzyme. In some embodiments, the compound modulates multipleenzymes, although it might modulate one enzyme to a greater extent thananother. Using the standard enzyme activity assays described herein, theactivity of the compounds could be characterized. In one embodiment, acompound exhibits an irreversible inhibition or activation of aparticular enzyme. In some embodiments, a compound reversibly inhibitsor activates an enzyme. In some embodiments, a compound alters thekinetics of the enzyme.

In one embodiment, for example, evaluating the interaction between thetest compound and host target enzyme includes one or more of (i)evaluating binding of the test compound to the enzyme; (ii) evaluating abiological activity of the enzyme; (iii) evaluating an enzymaticactivity (e.g., elongase activity) of the enzyme in the presence andabsence of test compound. The in vitro contacting can include forming areaction mixture that includes the test compound, enzyme, any requiredcofactor (e.g., biotin) or energy source (e.g., ATP, or radiolabeledATP), a substrate (e.g., acetyl-CoA, a sugar, a polypeptide, anucleoside, or any other metabolite, with or without label) andevaluating conversion of the substrate into a product. Evaluatingproduct formation can include, for example, detecting the transfer ofcarbons or phosphate (e.g., chemically or using a label, e.g., aradiolabel), detecting the reaction product, detecting a secondaryreaction dependent on the first reaction, or detecting a physicalproperty of the substrate, e.g., a change in molecular weight, charge,or pI.

Target enzymes for use in screening assays can be purified from anatural source, e.g., cells, tissues or organs comprising adipocytes(e.g., adipose tissue), liver, etc. Alternatively, target enzymes can beexpressed in any of a number of different recombinant DNA expressionsystems and can be obtained in large amounts and tested for biologicalactivity. For expression in recombinant bacterial cells, for example E.coli, cells are grown in any of a number of suitable media, for exampleLB, and the expression of the recombinant polypeptide induced by addingIPTG to the media or switching incubation to a higher temperature. Afterculturing the bacteria for a further period of between 2 and 24 hours,the cells are collected by centrifugation and washed to remove residualmedia. The bacterial cells are then lysed, for example, by disruption ina cell homogenizer and centrifuged to separate the dense inclusionbodies and cell membranes from the soluble cell components. Thiscentrifugation can be performed under conditions whereby the denseinclusion bodies are selectively enriched by incorporation of sugarssuch as sucrose into the buffer and centrifugation at a selective speed.If the recombinant polypeptide is expressed in the inclusion, these canbe washed in any of several solutions to remove some of thecontaminating host proteins, then solubilized in solutions containinghigh concentrations of urea (e.g., 8 M) or chaotropic agents such asguanidine hydrochloride in the presence of reducing agents such asbeta-mercaptoethanol or DTT (dithiothreitol). At this stage it may beadvantageous to incubate the polypeptide for several hours underconditions suitable for the polypeptide to undergo a refolding processinto a conformation which more closely resembles that of the nativepolypeptide. Such conditions generally include low polypeptide(concentrations less than 500 mg/ml), low levels of reducing agent,concentrations of urea less than 2 M and often the presence of reagentssuch as a mixture of reduced and oxidized glutathione which facilitatethe interchange of disulphide bonds within the protein molecule. Therefolding process can be monitored, for example, by SDS-PAGE or withantibodies which are specific for the native molecule. Followingrefolding, the polypeptide can then be purified further and separatedfrom the refolding mixture by chromatography on any of several supportsincluding ion exchange resins, gel permeation resins or on a variety ofaffinity columns.

Isolation and purification of host cell expressed polypeptide, orfragments thereof may be carried out by conventional means including,but not limited to, preparative chromatography and immunologicalseparations involving monoclonal or polyclonal antibodies.

These polypeptides may be produced in a variety of ways, including viarecombinant DNA techniques, to enable large scale production of pure,biologically active target enzyme useful for screening compounds for thepurposes of the invention. Alternatively, the target enzyme to bescreened could be partially purified or tested in a cellular lysate orother solution or mixture.

Target enzyme activity assays are preferably in vitro assays using theenzymes in solution or using cell or cell lysates that express suchenzymes, but the invention is not to be so limited. In certainembodiments, the enzyme is in solution. In other embodiments, the enzymeis associated with microsomes or in detergent. In other embodiments, theenzyme is immobilized to a solid or gel support. In certain embodiments,the enzyme is labeled to facilitate purification and/or detection. Inother embodiments, a substrate is labeled to facilitate purification andor detection. Labels include polypeptide tags, biotin, radiolabels,fluorescent labels, or a colorimetric label. Any art-accepted assay totest the activity of metabolic enzymes can be used in the practice ofthis invention. Preferably, many compounds are screened against multipletargets with high throughput screening assays.

Substrate and product levels can be evaluated in an in vitro system,e.g., in a biochemical extract, e.g., of proteins. For example, theextract may include all soluble proteins or a subset of proteins (e.g.,a 70% or 50% ammonium sulfate cut), the useful subset of proteinsdefined as the subset that includes the target enzyme. The effect of atest compound can be evaluated, for example, by measuring substrate andproduct levels at the beginning of a time course, and then comparingsuch levels after a predetermined time (e.g., 0.5, 1, or 2 hours) in areaction that includes the test compound and in a parallel controlreaction that does not include the test compound. This is one method fordetermining the effect of a test compound on the substrate-to-productratio in vitro. Reaction rates can obtained by linear regressionanalysis of radioactivity or other label incorporated vs. reaction timefor each incubation. K_(M) and V_(max) values can be determined bynon-linear regression analysis of initial velocities, according to thestandard Henri-Michaelis-Menten equation. k_(cat) can be obtained bydividing V_(max) values by reaction concentrations of enzyme, e.g.,derived by colorimetric protein determinations (e.g., Bio-RAD proteinassay, Bradford assay, Lowry method). In one embodiment, the compoundirreversibly inactivates the target enzyme. In another embodiment, thecompound reversibly inhibits the target enzyme. In some embodiments, thecompound reversibly inhibits the target enzyme by competitiveinhibition. In some embodiments, the compound reversibly inhibits thetarget enzyme by noncompetitive inhibition. In some embodiments, thecompound reversibly inhibits the target enzyme by uncompetitiveinhibition. In a further embodiment, the compound inhibits the targetenzyme by mixed inhibition. The mechanism of inhibition by the compoundcan be determined by standard assays known by those of ordinary skill inthe art.

Methods for the quantitative measurement of enzyme activity utilizing aphase partition system are described in U.S. Pat. No. 6,994,956, whichis incorporated by reference herein in its entirety. Specifically, aradiolabeled substrate and the product of the reaction aredifferentially partitioned into an aqueous phase and an immisciblescintillation fluid-containing organic phase, and enzyme activity isassessed either by incorporation of a radiolabeled-containingorganic-soluble moiety into product molecules (gain of signal assay) orloss of a radiolabel-containing organic-soluble moiety from substratemolecules (loss of signal assay). Scintillations are only detected whenthe radionuclide is in the organic, scintillant-contaning phase. Suchmethods can be employed to test the ability of a compound to inhibit theactivity of a target enzyme.

Cellular assays may be employed. An exemplary cellular assay includescontacting a test compound to a culture cell (e.g., a mammalian culturecell, e.g., a human culture cell) and then evaluating substrate andproduct levels in the cell, e.g., using any method described herein,such as Reverse Phase HPLC, LC-MS, or LC-MS/MS.

Substrate and product levels can be evaluated, e.g., by NMR, HPLC (See,e.g., Bak, M. I., and Ingwall, J. S. (1994) J. Clin. Invest. 93, 40-49),mass spectrometry, thin layer chromatography, or the use of radiolabeledcomponents (e.g., radiolabeled ATP for a kinase assay). For example, ³¹PNMR can be used to evaluate ATP and AMP levels. In one implementation,cells and/or tissue can be placed in a 10-mm NMR sample tube andinserted into a 1H/31P double-tuned probe situated in a 9.4-Teslasuperconducting magnet with a bore of 89 cm. If desired, cells can becontacted with a substance that provides a distinctive peak in order toindex the scans. Six ³¹P NMR spectra—each obtained by signal averagingof 104 free induction decays—can be collected using a 60° flip angle,15-microsecond pulse, 2.14-second delay, 6,000 Hz sweep width, and 2048data points using a GE-400 Omega NMR spectrometer (Bruker Instruments,Freemont, Calif., USA). Spectra are analyzed using 20-Hz exponentialmultiplication and zero- and first-order phase corrections. Theresonance peak areas can be fitted by Lorentzian line shapes using NMR1software (New Methods Research Inc., Syracuse, N.Y., USA). By comparingthe peak areas of fully relaxed spectra (recycle time: 15 seconds) andpartially saturated spectra (recycle time: 2.14 seconds), the correctionfactor for saturation can be calculated for the peaks. Peak areas can benormalized to cell and/or tissue weight or number and expressed inarbitrary area units. Another method for evaluating, e.g., ATP and AMPlevels includes lysing cells in a sample to form an extract, andseparating the extract by Reversed Phase HPLC, while monitoringabsorbance at 260 nm.

Another type of in vitro assay evaluates the ability of a test compoundto modulate interaction between a first enzyme pathway component and asecond enzyme pathway component This type of assay can be accomplished,for example, by coupling one of the components with a radioisotope orenzymatic label such that binding of the labeled component to the secondpathway component can be determined by detecting the labeled compound ina complex. An enzyme pathway component can be labeled with ¹²⁵I, ³⁵S,¹⁴C, or ³H, either directly or indirectly, and the radioisotope detectedby direct counting of radio-emission or by scintillation counting.Alternatively, a component can be enzymatically labeled with, forexample, horseradish peroxidase, alkaline phosphatase, or luciferase,and the enzymatic label detected by determination of conversion of anappropriate substrate to product. Competition assays can also be used toevaluate a physical interaction between a test compound and a target.

Soluble and/or membrane-bound forms of isolated proteins (e.g., enzymepathway components and their receptors or biologically active portionsthereof) can be used in the cell-free assays of the invention. Whenmembrane-bound forms of the enzyme are used, it may be desirable toutilize a solubilizing agent. Examples of such solubilizing agentsinclude non-ionic detergents such as n-octylglucoside,n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide,decanoyl-N-methylglucamide, Triton X-100, Triton X-114, Thesit,Isotridecypoly(ethylene glycol ether)n,3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate. Inanother example, the enzyme pathway component can reside in a membrane,e.g., a liposome or other vesicle.

Cell-free assays involve preparing a reaction mixture of the targetenzyme and the test compound under conditions and for a time sufficientto allow the two components to interact and bind, thus forming a complexthat can be removed and/or detected. In one embodiment, the targetenzyme is mixed with a solution containing one or more, and often manyhundreds or thousands, of test compounds. The target enzyme, includingany bound test compounds, is then isolated from unbound (i.e., free)test compounds, e.g., by size exclusion chromatography or affinitychromoatography. The test compound(s) bound to the target can then beseparated from the target enzyme, e.g., by denaturing the enzyme inorganic solvent, and the compounds identified by appropriate analyticalapproaches, e.g., LC-MS/MS.

The interaction between two molecules, e.g., target enzyme and testcompound, can also be detected, e.g., using a fluorescence assay inwhich at least one molecule is fluorescently labeled, e.g., to evaluatean interaction between a test compound and a target enzyme. One exampleof such an assay includes fluorescence energy transfer (FET or FRET forfluorescence resonance energy transfer) (See, for example, Lakowicz etal., U.S. Pat. No. 5,631,169; Stavrianopoulos, et al., U.S. Pat. No.4,868,103). A fluorophore label on the first, “donor” molecule isselected such that its emitted fluorescent energy will be absorbed by afluorescent label on a second, “acceptor” molecule, which in turn isable to fluoresce due to the absorbed energy. Alternately, aproteinaceous “donor” molecule may simply utilize the naturalfluorescent energy of tryptophan residues. Labels are chosen that emitdifferent wavelengths of light, such that the “acceptor” molecule labelmay be differentiated from that of the “donor.” Since the efficiency ofenergy transfer between the labels is related to the distance separatingthe molecules, the spatial relationship between the molecules can beassessed. In a situation in which binding occurs between the molecules,the fluorescent emission of the “acceptor” molecule label in the assayshould be maximal. A FET binding event can be conveniently measuredthrough standard fluorometric detection means well known in the art(e.g., using a fluorimeter).

Another example of a fluorescence assay is fluorescence polarization(FP). For FP, only one component needs to be labeled. A bindinginteraction is detected by a change in molecular size of the labeledcomponent. The size change alters the tumbling rate of the component insolution and is detected as a change in FP. See, e.g., Nasir et al.(1999) Comb Chem HTS 2:177-190; Jameson et al. (1995) Methods Enzymol246:283; See Anal Biochem. 255:257 (1998). Fluorescence polarization canbe monitored in multi-well plates. See, e.g., Parker et al. (2000)Journal of Biomolecular Screening 5:77-88; and Shoeman, et al. (1999)38, 16802-16809.

In another embodiment, determining the ability of the target enzyme tobind to a target molecule can be accomplished using real-timeBiomolecular Interaction Analysis (BIA) (See, e.g., Sjolander, S. andUrbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995)Curr. Opin. Struct. Biol. 5:699-705). “Surface plasmon resonance” or“BIA” detects biospecific interactions in real time, without labelingany of the interactants (e.g., BIAcore). Changes in the mass at thebinding surface (indicative of a binding event) result in alterations ofthe refractive index of light near the surface (the optical phenomenonof surface plasmon resonance (SPR)), resulting in a detectable signalwhich can be used as an indication of real-time reactions betweenbiological molecules.

In one embodiment, the target enzyme is anchored onto a solid phase. Thetarget enzyme/test compound complexes anchored on the solid phase can bedetected at the end of the reaction, e.g., the binding reaction. Forexample, the target enzyme can be anchored onto a solid surface, and thetest compound (which is not anchored), can be labeled, either directlyor indirectly, with detectable labels discussed herein.

It may be desirable to immobilize either the target enzyme or ananti-target enzyme antibody to facilitate separation of complexed fromuncomplexed forms of one or both of the proteins, as well as toaccommodate automation of the assay. Binding of a test compound totarget enzyme, or interaction of a target enzyme with a second componentin the presence and absence of a candidate compound, can be accomplishedin any vessel suitable for containing the reactants. Examples of suchvessels include microtiter plates, test tubes, and micro-centrifugetubes. In one embodiment, a fusion protein can be provided which adds adomain that allows one or both of the proteins to be bound to a matrix.For example, glutathione-S-transferase/target enzyme fusion proteins canbe adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo., USA) or glutathione derivatized microtiter plates, which are thencombined with the test compound or the test compound and either thenon-adsorbed target enzyme, and the mixture incubated under conditionsconducive to complex formation (e.g., at physiological conditions forsalt and pH). Following incubation, the beads or microtiter plate wellsare washed to remove any unbound components, the matrix immobilized inthe case of beads, and the complex determined either directly orindirectly, for example, as described above. Alternatively, thecomplexes can be dissociated from the matrix, and the level of targetenzyme binding or activity is determined using standard techniques.

Other techniques for immobilizing either a target enzyme or a testcompound on matrices include using conjugation of biotin andstreptavidin. Biotinylated target enzyme or test compounds can beprepared from biotin-NHS (N-hydroxy-succinimide) using techniques knownin the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.),and immobilized in the wells of streptavidin-coated 96 well plates(Pierce Chemical).

In order to conduct the assay, the non-immobilized component is added tothe coated surface containing the anchored component. After the reactionis complete, unreacted components are removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized on thesolid surface. The detection of complexes anchored on the solid surfacecan be accomplished in a number of ways. Where the previouslynon-immobilized component is pre-labeled, the detection of labelimmobilized on the surface indicates that complexes were formed. Wherethe previously non-immobilized component is not pre-labeled, an indirectlabel can be used to detect complexes anchored on the surface, e.g.,using a labeled antibody specific for the immobilized component (theantibody, in turn, can be directly labeled or indirectly labeled with,e.g., a labeled anti-Ig antibody).

In one embodiment, this assay is performed utilizing antibodies reactivewith a target enzyme but which do not interfere with binding of thetarget enzyme to the test compound and/or substrate. Such antibodies canbe derivatized to the wells of the plate, and unbound target enzymetrapped in the wells by antibody conjugation. Methods for detecting suchcomplexes, in addition to those described above for the GST-immobilizedcomplexes, include immunodetection of complexes using antibodiesreactive with the target enzyme, as well as enzyme-linked assays whichrely on detecting an enzymatic activity associated with the targetenzyme.

Alternatively, cell free assays can be conducted in a liquid phase. Insuch an assay, the reaction products are separated from unreactedcomponents, by any of a number of standard techniques, including but notlimited to: differential centrifugation (See, for example, Rivas, G.,and Minton, A. P., (1993) Trends Biochem Sci 18:284-7); chromatography(gel filtration chromatography, ion-exchange chromatography);electrophoresis (See, e.g., Ausubel, F. et al., eds. Current Protocolsin Molecular Biology 1999, J. Wiley: New York); and immunoprecipitation(See, for example, Ausubel, F. et al., eds. (1999) Current Protocols inMolecular Biology, J. Wiley: New York). Such resins and chromatographictechniques are known to one skilled in the art (See, e.g., Heegaard, N.H., (1998) J Mol Recognit 11:141-8; Hage, D. S., and Tweed, S. A. (1997)J Chromatogr B Biomed Sci Appl. 699:499-525). Further, fluorescenceenergy transfer may also be conveniently utilized, as described herein,to detect binding without further purification of the complex fromsolution.

In a preferred embodiment, the assay includes contacting the targetenzyme or biologically active portion thereof with a known compoundwhich binds the target enzyme to form an assay mixture, contacting theassay mixture with a test compound, and determining the ability of thetest compound to interact with the target enzyme, wherein determiningthe ability of the test compound to interact with the target enzymeincludes determining the ability of the test compound to preferentiallybind to the target enzyme, or to modulate the activity of the targetenzyme, as compared to the known compound (e.g., a competition assay).In another embodiment, the ability of a test compound to bind to andmodulate the activity of the target enzyme is compared to that of aknown activator or inhibitor of such target enzyme.

The target enzymes of the invention can, in vivo, interact with one ormore cellular or extracellular macromolecules, such as proteins, whichare either heterologous to the host cell or endogenous to the host cell,and which may or may not be recombinantly expressed. For the purposes ofthis discussion, such cellular and extracellular macromolecules arereferred to herein as “binding partners.” Compounds that disrupt suchinteractions can be useful in regulating the activity of the targetenzyme. Such compounds can include, but are not limited to moleculessuch as antibodies, peptides, and small molecules. In an alternativeembodiment, the invention provides methods for determining the abilityof the test compound to modulate the activity of a target enzyme throughmodulation of the activity of a downstream effector of such targetenzyme. For example, the activity of the effector molecule on anappropriate target can be determined, or the binding of the effector toan appropriate target can be determined, as previously described.

To identify compounds that interfere with the interaction between thetarget enzyme and its cellular or extracellular binding partner(s), areaction mixture containing the target enzyme and the binding partner isprepared, under conditions and for a time sufficient, to allow the twoproducts to form a complex. In order to test an inhibitory compound, thereaction mixture is provided in the presence and absence of the testcompound. The test compound can be initially included in the reactionmixture, or can be added at a time subsequent to the addition of thetarget and its cellular or extracellular binding partner. Controlreaction mixtures are incubated without the test compound or with aplacebo. The formation of any complexes between the target product andthe cellular or extracellular binding partner is then detected. Theformation of a complex in the control reaction, but not in the reactionmixture containing the test compound, indicates that the compoundinterferes with the interaction of the target product and theinteractive binding partner. Additionally, complex formation withinreaction mixtures containing the test compound and normal target enzymecan also be compared to complex formation within reaction mixturescontaining the test compound and mutant target enzyme. This comparisoncan be important in those cases wherein it is desirable to identifycompounds that disrupt interactions of mutant but not normal targetenzymes.

The assays described herein can be conducted in a heterogeneous orhomogeneous format. Heterogeneous assays involve anchoring either thetarget enzyme or the binding partner, substrate, or tests compound ontoa solid phase, and detecting complexes anchored on the solid phase atthe end of the reaction. In homogeneous assays, the entire reaction iscarried out in a liquid phase. In either approach, the order of additionof reactants can be varied to obtain different information about thecompounds being tested. For example, test compounds that interfere withthe interaction between the target enzyme and a binding partners orsubstrate, e.g., by competition, can be identified by conducting thereaction in the presence of the test substance. Alternatively, testcompounds that disrupt preformed complexes, e.g., compounds with higherbinding constants that displace one of the components from the complex,can be tested by adding the test compound to the reaction mixture aftercomplexes have been formed. The various formats are briefly describedbelow.

In a heterogeneous assay system, either the target enzyme or theinteractive cellular or extracellular binding partner or substrate, isanchored onto a solid surface (e.g., a microtiter plate), while thenon-anchored species is labeled, either directly or indirectly. Theanchored species can be immobilized by non-covalent or covalentattachments. Alternatively, an immobilized antibody specific for thespecies to be anchored can be used to anchor the species to the solidsurface.

In order to conduct the assay, the partner of the immobilized species isexposed to the coated surface with or without the test compound. Afterthe reaction is complete, unreacted components are removed (e.g., bywashing) and any complexes formed will remain immobilized on the solidsurface. Where the non-immobilized species is pre-labeled, the detectionof label immobilized on the surface indicates that complexes wereformed. Where the non-immobilized species is not pre-labeled, anindirect label can be used to detect complexes anchored on the surface;e.g., using a labeled antibody specific for the initiallynon-immobilized species (the antibody, in turn, can be directly labeledor indirectly labeled with, e.g., a labeled anti-Ig antibody). Dependingupon the order of addition of reaction components, test compounds thatinhibit complex formation or that disrupt preformed complexes can bedetected.

Alternatively, the reaction can be conducted in a liquid phase in thepresence or absence of the test compound, the reaction productsseparated from unreacted components, and complexes detected; e.g., usingan immobilized antibody specific for one of the binding components toanchor any complexes formed in solution, and a labeled antibody specificfor the other partner to detect anchored complexes. Again, dependingupon the order of addition of reactants to the liquid phase, testcompounds that inhibit complex or that disrupt preformed complexes canbe identified.

In an alternate embodiment of the invention, a homogeneous assay can beused. For example, a preformed complex of the target enzyme and theinteractive cellular or extracellular binding partner product orsubstrate is prepared in that either the target enzyme or their bindingpartners or substrates are labeled, but the signal generated by thelabel is quenched due to complex formation (See, e.g., U.S. Pat. No.4,109,496 that utilizes this approach for immunoassays). The addition ofa test substance that competes with and displaces one of the speciesfrom the preformed complex will result in the generation of a signalabove background. In this way, test compounds that disrupt targetenzyme-binding partner or substrate contact can be identified.

In yet another aspect, the target enzyme can be used as “bait protein”in a two-hybrid assay or three-hybrid assay (See, e.g., U.S. Pat. No.5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J.Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent,International patent application Publication No. WO94/10300), toidentify other proteins that bind to or interact with target enzyme(“target enzyme binding protein” or “target enzyme-bp”) and are involvedin target enzyme pathway activity. Such target enzyme-bps can beactivators or inhibitors of the target enzyme or target enzyme targetsas, for example, downstream elements of the target enzyme pathway.

In another embodiment, modulators of a target enzyme's gene expressionare identified. For example, a cell or cell free mixture is contactedwith a candidate compound and the expression of the target enzyme mRNAor protein evaluated relative to the level of expression of targetenzyme mRNA or protein in the absence of the candidate compound. Whenexpression of the target enzyme component mRNA or protein is greater inthe presence of the candidate compound than in its absence, thecandidate compound is identified as a stimulator of target enzyme mRNAor protein expression. Alternatively, when expression of the targetenzyme mRNA or protein is less (statistically significantly less) in thepresence of the candidate compound than in its absence, the candidatecompound is identified as an inhibitor of the target enzyme mRNA orprotein expression. The level of the target enzyme mRNA or proteinexpression can be determined by methods for detecting target enzyme mRNAor protein, e.g., Westerns, Northerns, PCR, mass spectroscopy, 2-D gelelectrophoresis, and so forth, all which are known to those of ordinaryskill in the art.

Assays for producing enzyme targets, testing their activity, andconducting screens for their inhibition or activation are describedbelow using examples of enzymes related to fatty acid biosynthesis.These assays can be adapted by one of ordinary skill in the art, orother assays known in the art can be used, to test the activity of othertargets of the invention.

3.1 High Throughput Screening of Compounds and Target Enzymes

In one embodiment, high throughput screening using, e.g., massspectrometry can be used to screen a number of compounds and a number ofpotential target enzymes simultaneously. Mass spectrometry can beutilized for determination of metabolite levels and enzymatic activity.

The levels of specific metabolites (e.g. AMP, ATP) can be quantified byliquid chromatography-mass spectrometry (LC-MS/MS). A metabolite ofinterest will have a specific chromatographic retention time at whichpoint the mass spectrometer performs a selected reaction monitoring scanevent (SRM) that consists of three identifiers:

1) The metabolite's mass (the parent ion);

2) The energy required to fragment the parent ion in a collision withargon to yield a fragment with a specific mass; and

3) The mass of the specific fragment ion.

Utilizing the above identifiers, the accumulation of a metabolite can bemeasured whose production depends on the activity of a metabolic enzymeof interest. By adding an excess of enzyme substrate to a cellularlysate, so as to make the activity of the enzyme rate limiting, theaccumulation of enzymatic product over time is then measured by LC-MS/MSas outlined above, and serves as a function of the metabolic enzyme'sactivity. An example of such an assay is reported in Munger et al, 2006PLoS Pathogens, 2: 1-11, incorporated herein by reference in itsentirety, in which the activity of phosphofructokinase present ininfected lysates was measured by adding an excess of thephosphofructokinase substrates ATP and fructose phosphate and measuringfructose bisphosphate accumulation by LC-MS/MS. This approach can beadopted to measure the activities of numerous host target enzymes.

3.2 Kinetic Flux Profiling (KFP) to Assess Potential Antiviral Compounds

In a further embodiment of the invention, cellular metabolic fluxes areprofiled in the presence or absence of a virus using kinetic fluxprofiling (KFP) (See Munger et al. 2008 Nature Biotechnology, 26:1179-1186) in the presence or absence of a compound found to inhibit atarget enzyme in one of the aforementioned assays. Such metabolic fluxprofiling provides additional (i) guidance about which components of ahost's metabolism can be targeted for antiviral intervention; (ii)guidance about the metabolic pathways targeted by different viruses; and(iii) validation of compounds as potential antiviral agents based ontheir ability to offset the metabolic flux caused by a virus or triggercell-lethal metabolic derangements specifically in virally infectedcells. In one embodiment, the kinetic flux profiling methods of theinvention can be used for screening to determine (i) the specificalterations in metabolism caused by different viruses and (ii) theability of a compound to offset (or specifically augment) alterations inmetabolic flux caused by different viruses.

Thus, in one embodiment of the invention, cells are infected with avirus and metabolic flux is assayed at different time points after virusinfection, such time points known to one of skill in the art. Forexample, for HCMV, flux can be measured 24, 48, or 72 hourspost-infection, whereas for a faster growing virus like HSV, flux can bemeasured at 6, 12, or 18 hours post-infection. If the metabolic flux isaltered in the presence of the virus, then the virus alters cellularmetabolism during infection. The type of metabolic flux alterationobserved (See above and examples herein) will provide guidance as to thecellular pathways that the virus acts on. Assays well known to those ofskill in the art and described herein below can then be employed toconfirm the target of the virus. Similarly, compounds can be tested forthe ability to interfere with the virus in the assays for antiviralactivity described in Section 4 below. If it appears that a virusmodulates the level and/or activity of a particular enzyme, inhibitorsof that enzyme can be tested for their antiviral effect. Ifwell-characterized compounds are observed to be effective antivirals,other compounds that modulate the same target can similarly be assessedas potential antivirals.

In one embodiment of the invention, a virus infected cell is contactedwith a compound and metabolic flux is measured. If the metabolic flux inthe presence of the compound is different from the metabolic flux in theabsence of the compound, in a manner wherein the metabolic effects ofthe virus have been inhibited or augmented, then a compound thatmodulates the virus' ability to alter the metabolic flux has beenidentified. The type of metabolic flux alteration observed will provideguidance as to the cellular pathway that the compound is acting on.Assays well known to those of skill in the art and described herein canthen be employed to confirm the target of the antiviral compound.

In one embodiment, high throughput metabolome quantitation massspectrometry can be used to screen for changes in metabolism caused byinfection of a virus and whether or not a compound or library ofcompounds offsets these changes. See Munger et al. 2006. PLoS Pathogens,2: 1-11.

3.3 Identification of compounds

Using metabolome and fluxome-based analysis of virus infected cells, theinventors have identified host cell target enzymes listed in Table 1(Section 1) and demonstrated that virus replication can be reduced byreducing expression of the target enzymes. Further, any compound ofinterest can be tested for its ability to modulate the activity of theseenzymes. Alternatively, compounds can be tested for their ability toinhibit any other host cell enzyme related to metabolism. Once suchcompounds are identified as having metabolic enzyme-modulating activity,they can be further tested for their antiviral activity as described inSection 4. Alternatively, compounds can be screened for antiviralactivity and optionally characterized using the metabolic screeningassays described herein.

In one embodiment, high throughput screening methods are used to providea combinatorial chemical or peptide library (e.g., a publicly availablelibrary) containing a large number of potential therapeutic compounds(potential modulators or ligand compounds). Such “combinatorial chemicallibraries” or “ligand libraries” are then screened in one or moreassays, as described in Section 3 herein, to identify those librarymembers (particular chemical species or subclasses) that display adesired characteristic activity. The compounds thus identified can serveas conventional “lead compounds” or can themselves be used as potentialor actual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (See, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493(1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (e.g., PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT PublicationNo. WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomerssuch as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc.Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides(Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer.Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of smallcompound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)),oligocarbamates (Cho et al., Science 261:1303 (1993)), and/or peptidylphosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleicacid libraries (See Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (See, e.g., U.S. Pat. No. 5,539,083), antibodylibraries (See, e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314(1996) and PCT/US96/10287), carbohydrate libraries (See, e.g., Liang etal., Science, 274:1520-1522 (1996) and International Patent ApplicationPublication NO. WO 1997/000271), small organic molecule libraries (See,e.g., benzodiazepines, Baum C&EN, January 18, page 33 (1993);isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones andmetathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos.5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337;benzodiazepines, U.S. Pat. No. 5,288,514, and the like). Additionalexamples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and Gallop et al. (1994) J. Med. Chem. 37:1233.

Some exemplary libraries are used to generate variants from a particularlead compound. One method includes generating a combinatorial library inwhich one or more functional groups of the lead compound are varied,e.g., by derivatization. Thus, the combinatorial library can include aclass of compounds which have a common structural feature (e.g.,scaffold or framework). Devices for the preparation of combinatoriallibraries are commercially available (See, e.g., 357 MPS, 390 MPS,Advanced Chem Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass.,433A Applied Biosystems, Foster City, Calif., 9050 Plus, Millipore,Bedford, Mass.). In addition, numerous combinatorial libraries arethemselves commercially available (See, e.g., ComGenex, Princeton, N.J.,Asinex, Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar, Ltd, Moscow,RU, 3D Pharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md.,etc.). The test compounds can also be obtained from: biologicallibraries; peptoid libraries (libraries of molecules having thefunctionalities of peptides, but with a novel, non-peptide backbonewhich are resistant to enzymatic degradation but which neverthelessremain bioactive; See, e.g., Zuckermann, R. N. et al. (1994) J. Med.Chem. 37:2678-85); spatially addressable parallel solid phase orsolution phase libraries; synthetic library methods requiringdeconvolution; the ‘one-bead one-compound’ library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibraries include libraries of nucleic acids and libraries of proteins.Some nucleic acid libraries encode a diverse set of proteins (e.g.,natural and artificial proteins; others provide, for example, functionalRNA and DNA molecules such as nucleic acid aptamers or ribozymes. Apeptoid library can be made to include structures similar to a peptidelibrary. (See also Lam (1997) Anticancer Drug Des. 12:145). A library ofproteins may be produced by an expression library or a display library(e.g., a phage display library). Libraries of compounds may be presentedin solution (e.g., Houghten (1992) Biotechniques 13:412-421), or onbeads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature364:555-556), bacteria (Ladner, U.S. Pat. No. 5,223,409), spores (LadnerU.S. Pat. No. 5,223,409), plasmids (Cull et al. (1992) Proc Natl AcadSci USA 89:1865-1869) or on phage (Scott and Smith (1990) Science249:386-390; Devlin (1990) Science 249:404-406; Cwirla et al. (1990)Proc. Natl. Acad. Sci. 87:6378-6382; Felici (1991) J. Mol. Biol.222:301-310; Ladner supra.). Enzymes can be screened for identifyingcompounds which can be selected from a combinatorial chemical library orany other suitable source (Hogan, Jr., Nat. Biotechnology 15:328, 1997).

Any assay herein, e.g., an in vitro assay or an in vivo assay, can beperformed individually, e.g., just with the test compound, or withappropriate controls. For example, a parallel assay without the testcompound, or other parallel assays without other reaction components,e.g., without a target or without a substrate. Alternatively, it ispossible to compare assay results to a reference, e.g., a referencevalue, e.g., obtained from the literature, a prior assay, and so forth.Appropriate correlations and art known statistical methods can be usedto evaluate an assay result. See Section 3.1 above.

Once a compound is identified as having a desired effect, productionquantities of the compound can be synthesized, e.g., producing at least50 mg, 500 mg, 5 g, or 500 g of the compound. Although a compound thatis able to penetrate a host cell is preferable in the practice of theinvention, a compound may be combined with solubilizing agents oradministered in combination with another compound or compounds tomaintain its solubility, or help it enter a host cell, e.g., by mixturewith lipids. The compound can be formulated, e.g., for administration toa subject, and may also be administered to the subject.

4. Characterization of Antiviral Activity of Compounds

4.1 Viruses

The present invention provides compounds for use in the prevention,management and/or treatment of viral infection. The antiviral activityof compounds against any virus can be tested using techniques describedin Section 4.2 herein below. The virus may be enveloped or naked, have aDNA or RNA genome, or have a double-stranded or single-stranded genome.See, e.g., FIG. 1 modified from Flint et al., Principles of Virology:Molecular Biology, Pathogenesis and Control of Animal Viruses. 2ndedition, ASM Press, 2003, for a subset of virus families and theirclassification, as well as a subset of viruses against which compoundscan be assessed for antiviral activity. In specific embodiments, thevirus infects human. In other embodiments, the virus infects non-humananimals. In a specific embodiment, the virus infects pigs, fowl, otherlivestock, or pets.

In certain embodiments, the virus is an enveloped virus. Envelopedviruses include, but are not limited to viruses that are members of thehepadnavirus family, herpesvirus family, iridovirus family, poxvirusfamily, flavivirus family, togavirus family, retrovirus family,coronavirus family, filovirus family, rhabdovirus family, bunyavirusfamily, orthomyxovirus family, paramyxovirus family, and arenavirusfamily. Non-limiting examples of viruses that belong to these familiesare included in Table 3.

TABLE 3 Families of Enveloped Viruses Virus Family Members Hepadnavirushepatitis B virus (HBV), woodchuck hepatitis virus, ground squirrel(Hepadnaviridae) hepatitis virus, duck hepatitis B virus, heronhepatitis B virus Herpesvirus herpes simplex virus (HSV) types 1 and 2,varicella-zoster virus, (Herpesviridae) cytomegalovirus (CMV), humancytomegalovirus (HCMV), Epstein- Barr virus (EBV), human herpesvirus 6(variants A and B), human herpesvirus 7, human herpesvirus 8, Kaposi'ssarcoma-associated herpes virus (KSHV), B virus Poxvirus vaccinia virus,variola virus, smallpox virus, monkeypox virus, (Poxviridae) cowpoxvirus, camelpox virus, mousepox virus, raccoonpox viruses, molluscumcontagiosum virus, orf virus, milker's nodes virus, bovin papullarstomatitis virus, sheeppox virus, goatpox virus, lumpy skin diseasevirus, fowlpox virus, canarypox virus, pigeonpox virus, sparrowpoxvirus, myxoma virus, hare fibroma virus, rabbit fibroma virus, squirrelfibroma viruses, swinepox virus, tanapox virus, Yabapox virus Flavivirusdengue virus, hepatitis C virus (HCV), GB hepatitis viruses (GBV-A,(Flaviviridae) GBV-B and GBV-C), West Nile virus, yellow fever virus,St.Louis encephalitis virus, Japanese encephalitis virus, Powassanvirus, tick- borne encephalitis virus, Kyasanur Forest disease virusTogavirus Venezuelan equine encephalitis virus, chikungunya virus, RossRiver (Togaviridae) virus, Mayaro virus, Sindbis virus, rubella virusRetrovirus human immunodeficiency virus (HIV) types 1 and 2, human Tcell (Retroviridae) leukemia virus (HTLV) types 1, 2, and 5, mousemammary tumor virus (MMTV), Rous sarcoma virus (RSV), lentivirusesCoronavirus severe acute respiratory syndrome (SARS) virus(Coronaviridae) Filovirus Ebola virus, Marburg virus (Filoviridae)Rhabdovirus rabies virus, vesicular stomatitis virus (Rhabdoviridae)Bunyavirus Crimean-Congo hemorrhagic fever virus, Rift Valley fevervirus, La (Bunyaviridae) Crosse virus, Hantaan virus Orthomyxovirusinfluenza virus (types A, B, and C) (Orthomyxoviridae) Paramyxovirusparainfluenza virus, respiratory syncytial virus (types A and B),(Paramyxoviridae) measles virus, mumps virus Arenavirus lymphocyticchoriomeningitis virus, Junin virus, Machupo virus, (Arenaviridae)Guanarito virus, Lassa virus, Ampari virus, Flexal virus, Ippy virus,Mobala virus, Mopeia virus, Latino virus, Parana virus, Pichinde virus,Tacaribe virus, Tamiami virus

In some embodiments, the virus is a non-enveloped virus, i.e., the virusdoes not have an envelope and is naked. Non-limiting examples of suchviruses include viruses that are members of the parvovirus family,circovirus family, polyoma virus family, papillomavirus family,adenovirus family, iridovirus family, reovirus family, birnavirusfamily, calicivirus family, and picornavirus family. Examples of virusesthat belong to these families include, but are not limited to, those setforth in Table 4.

TABLE 4 Families of Non-Enveloped (Naked) Viruses Virus Family MembersParvovirus canine parvovirus, parvovirus B19 (Parvoviridae) Circovirusporcine circovirus type 1 and 2, BFDV (Beak and Feather Disease(Circoviridae) Virus), chicken anaemia virus Polyomavirus simian virus40 (SV40), JC virus, BK virus, Budgerigar fledgling (Polyomaviridae)disease virus Papillomavirus human papillomavirus, bovine papillomavirus(BPV) type 1 (Papillomaviridae) Adenovirus human adenovirus (HAdV-A,HAdV-B, HAdV-C, HAdV-D, HAdV-E, (Adenoviridae) and HAdV-F), fowladenovirus A, ovine adenovirus D, frog adenovirus Reovirus humanorbivirus, human coltivirus, mammalian orthoreovirus, (Reoviridae)bluetongue virus, rotavirus A, rotaviruses (groups B to G), Coloradotick fever virus, aquareovirus A, cypovirus 1, Fiji disease virus, ricedwarf virus, rice ragged stunt virus, idnoreovirus 1, mycoreovirus 1Birnavirus bursal disease virus, pancreatic necrosis virus(Birnaviridae) Calicivirus swine vesicular exanthema virus, rabbithemorrhagic disease virus, (Caliciviridae) Norwalk virus, Sapporo virusPicornavirus human polioviruses (1-3), human coxsackieviruses A1-22, 24(CA1-22 (Picornaviridae) and CA24, CA23 = echovirus 9), humancoxsackieviruses (B1-6 (CB1-6)), human echoviruses 1-7, 9, 11-27, 29-33,vilyuish virus, simian enteroviruses 1-18 (SEV1-18), porcineenteroviruses 1-11 (PEV1-11), bovine enteroviruses 1-2 (BEV1-2),hepatitis A virus, rhinoviruses, hepatoviruses, cardioviruses,aphthoviruses, echoviruses

In certain embodiments, the virus is a DNA virus. In other embodiments,the virus is a RNA virus. In one embodiment, the virus is a DNA or a RNAvirus with a single-stranded genome. In another embodiment, the virus isa DNA or a RNA virus with a double-stranded genome.

In some embodiments, the virus has a linear genome. In otherembodiments, the virus has a circular genome. In some embodiments, thevirus has a segmented genome. In other embodiments, the virus has anon-segmented genome.

In some embodiments, the virus is a positive-stranded RNA virus. Inother embodiments, the virus is a negative-stranded RNA virus. In oneembodiment, the virus is a segmented, negative-stranded RNA virus. Inanother embodiment, the virus is a non-segmented negative-stranded RNAvirus.

In some embodiments, the virus is an icosahedral virus. In otherembodiments, the virus is a helical virus. In yet other embodiments, thevirus is a complex virus.

In certain embodiments, the virus is a herpes virus, e.g., HSV-1, HSV-2,and CMV. In other embodiments, the virus is not a herpes virus (e.g.,HSV-1, HSV-2, and CMV). In a specific embodiment, the virus is HSV. Inan alternative embodiment, the virus is not HSV. In another embodiment,the virus is HCMV. In a further alternative embodiment, the virus is notHCMV. In another embodiment, the virus is a liver trophic virus. In analternative embodiment, the virus is not a liver trophic virus. Inanother embodiment, the virus is a hepatitis virus. In an alternateembodiment, the virus is not a hepatitis virus. In another embodiment,the virus is a hepatitis C virus. In a further alternative embodiment,the virus is not a hepatitis C virus. In another specific embodiment,the virus is an influenza virus. In an alternative embodiment, the virusis not an influenza virus. In some embodiments, the virus is aretrovirus. In some embodiments, the virus is not a retrovirus. In someembodiments, the virus is HIV. In other embodiments, the virus is notHIV. In certain embodiments, the virus is a hepatitis B virus. Inanother alternative embodiment, the virus is not a hepatitis B virus. Ina specific embodiment, the virus is EBV. In a specific alternativeembodiment, the virus is not EBV. In some embodiments, the virus isKaposi's sarcoma-associated herpes virus (KSHV). In some alternativeembodiments, the virus is not KSHV. In certain embodiments the virus isa variola virus. In certain alternative embodiments, the virus is notvariola virus. In one embodiment, the virus is a Dengue virus. In onealternative embodiment, the virus is not a Dengue virus. In otherembodiments, the virus is a SARS virus. In other alternativeembodiments, the virus is not a SARS virus. In a specific embodiment,the virus is an Ebola virus. In an alternative embodiment, the virus isnot an Ebola virus. In some embodiments the virus is a Marburg virus. Inan alternative embodiment, the virus is not a Marburg virus. In certainembodiments, the virus is a measles virus. In some alternativeembodiments, the virus is not a measles virus. In particularembodiments, the virus is a vaccinia virus. In alternative embodiments,the virus is not a vaccinia virus. In some embodiments, the virus isvaricella-zoster virus (VZV). In an alternative embodiment the virus isnot VZV. In some embodiments, the virus is a picornavirus. Inalternative embodiments, the virus is not a picornavirus. In certainembodiments the virus is not a rhinovirus. In certain embodiments, thevirus is a poliovirus. In alternative embodiments, the virus is not apoliovirus. In some embodiments, the virus is an adenovirus. Inalternative embodiments, the virus is not adenovirus. In particularembodiments, the virus is a coxsackievirus (e.g., coxsackievirus B3). Inother embodiments, the virus is not a coxsackievirus (e.g.,coxsackievirus B3). In some embodiments, the virus is a rhinovirus. Inother embodiments, the virus is not a rhinovirus. In certainembodiments, the virus is a human papillomavirus (HPV). In otherembodiments, the virus is not a human papillomavirus. In certainembodiments, the virus is a virus selected from the group consisting ofthe viruses listed in Tables 3 and 4. In other embodiments, the virus isnot a virus selected from the group consisting of the viruses listed inTables 3 and 4. In one embodiment, the virus is not one or more virusesselected from the group consisting of the viruses listed in Tables 3 and4.

The antiviral activities of compounds against any type, subtype orstrain of virus can be assessed. For example, the antiviral activity ofcompounds against naturally occurring strains, variants or mutants,mutagenized viruses, reassortants and/or genetically engineered virusescan be assessed.

The lethality of certain viruses, the safety issues concerning workingwith certain viruses and/or the difficulty in working with certainviruses may preclude (at least initially) the characterization of theantiviral activity of compounds on such viruses. Under suchcircumstances, other animal viruses that are representative of suchviruses may be utilized. For example, SIV may be used initially tocharacterize the antiviral activity of compounds against HIV. Further,Pichinde virus may be used initially to characterize the antiviralactivity of compounds against Lassa fever virus.

In some embodiments, the virus achieves peak titer in cell culture or asubject in 4 hours or less, 6 hours or less, 8 hours or less, 12 hoursor less, 16 hours or less, or 24 hours or less. In other embodiments,the virus achieves peak titers in cell culture or a subject in 48 hoursor less, 72 hours or less, or 1 week or less. In other embodiments, thevirus achieves peak titers after about more than 1 week. In accordancewith these embodiments, the viral titer may be measured in the infectedtissue or serum.

In some embodiments, the virus achieves in cell culture a viral titer of10⁴ pfu/ml or more, 5×10⁴ pfu/ml or more, 10⁵ pfu/ml or more, 5×10⁵pfu/ml or more, 10⁶ pfu/ml or more, 5×10⁶ pfu/ml or more, 10⁷ pfu/ml ormore, 5×10⁷ pfu/ml or more, 10⁸ pfu/ml or more, 5×10⁸ pfu/ml or more,10⁹ pfu/ml or more, 5×10⁹ pfu/ml or more, or 10¹⁰ pfu/ml or more. Incertain embodiments, the virus achieves in cell culture a viral titer of10⁴ pfu/ml or more, 5×10⁴ pfu/ml or more, 10⁵ pfu/ml or more, 5×10⁵pfu/ml or more, 10⁶ pfu/ml or more, 5×10⁶ pfu/ml or more, 10⁷ pfu/ml ormore, 5×10⁷ pfu/ml or more, 10⁸ pfu/ml or more, 5×10⁸ pfu/ml or more,10⁹ pfu/ml or more, 5×10⁹ pfu/ml or more, or 10¹⁰ pfu/ml or more within4 hours, 6 hours, 8 hours, 12 hours, 16 hours, or 24 hours or less. Inother embodiments, the virus achieves in cell culture a viral titer of10⁴ pfu/ml or more, 5×10⁴ pfu/ml or more, 10⁵ pfu/ml or more, 5×10⁵pfu/ml or more, 10⁶ pfu/ml or more, 5×10⁶ pfu/ml or more, 10⁷ pfu/ml ormore, 5×10⁷ pfu/ml or more, 10⁸ pfu/ml or more, 5×10⁸ pfu/ml or more,10⁹ pfu/ml or more, 5×10⁹ pfu/ml or more, or 10¹⁰ pfu/ml or more within48 hours, 72 hours, or 1 week.

In some embodiments, the virus achieves a viral yield of 1 pfu/ml ormore, 10 pfu/ml or more, 5×10¹ pfu/ml or more, 10² pfu/ml or more, 5×10²pfu/ml or more, 10³ pfu/ml or more, 2.5×10³ pfu/ml or more, 5×10³ pfu/mlor more, 10⁴ pfu/ml or more, 2.5×10⁴ pfu/ml or more, 5×10⁴ pfu/ml ormore, or 10⁵ pfu/ml or more in a subject. In certain embodiments, thevirus achieves a viral yield of 1 pfu/ml or more, 10 pfu/ml or more,5×10¹ pfu/ml or more, 10² pfu/ml or more, 5×10² pfu/ml or more, 10³pfu/ml or more, 2.5×10³ pfu/ml or more, 5×10³ pfu/ml or more, 10⁴ pfu/mlor more, 2.5×10⁴ pfu/ml or more, 5×10⁴ pfu/ml or more, or 10⁵ pfu/ml ormore in a subject within 4 hours, 6 hours, 8 hours, 12 hours, 16 hours,24 hours, or 48 hours. In certain embodiments, the virus achieves aviral yield of 1 pfu/ml or more, 10 pfu/ml or more, 10¹ pfu/ml or more,5×10¹ pfu/ml or more, 10² pfu/ml or more, 5×10² pfu/ml or more, 10³pfu/ml or more, 2.5×10³ pfu/ml or more, 5×10³ pfu/ml or more, 10⁴ pfu/mlor more, 2.5×10⁴ pfu/ml or more, 5×10⁴ pfu/ml or more, or 10⁵ pfu/ml ormore in a subject within 48 hours, 72 hours, or 1 week. In accordancewith these embodiments, the viral yield may be measured in the infectedtissue or serum. In a specific embodiment, the subject isimmunocompetent. In another embodiment, the subject is immunocompromisedor immunosuppressed.

In some embodiments, the virus achieves a viral yield of 1 pfu or more,10 pfu or more, 5×10¹ pfu or more, 10² pfu or more, 5×10² pfu or more,10³ pfu or more, 2.5×10³ pfu or more, 5×10³ pfu or more, 10⁴ pfu ormore, 2.5×10⁴ pfu or more, 5×10⁴ pfu or more, or 10⁵ pfu or more in asubject. In certain embodiments, the virus achieves a viral yield of 1pfu or more, 10 pfu or more, 5×10¹ pfu or more, 10² pfu or more, 5×10²pfu or more, 10³ pfu or more, 2.5×10³ pfu or more, 5×10³ pfu or more,10⁴ pfu or more, 2.5×10⁴ pfu or more, 5×10⁴ pfu or more, or 10⁵ pfu ormore in a subject within 4 hours, 6 hours, 8 hours, 12 hours, 16 hours,24 hours, or 48 hours. In certain embodiments, the virus achieves aviral yield of 1 pfu or more, 10 pfu or more, 10¹ pfu or more, 5×10¹ pfuor more, 10² pfu or more, 5×10² pfu or more, 10³ pfu or more, 2.5×10³pfu or more, 5×10³ pfu or more, 10⁴ pfu or more, 2.5×10⁴ pfu or more,5×10⁴ pfu or more, or 10⁵ pfu or more in a subject within 48 hours, 72hours, or 1 week. In accordance with these embodiments, the viral yieldmay be measured in the infected tissue or serum. In a specificembodiment, the subject is immunocompetent. In another embodiment, thesubject is immunocompromised or immunosuppressed.

In some embodiments, the virus achieves a viral yield of 1 infectiousunit or more, 10 infectious units or more, 5×10¹ infectious units ormore, 10² infectious units or more, 5×10² infectious units or more, 10³infectious units or more, 2.5×10³ infectious units or more, 5×10³infectious units or more, 10⁴ infectious units or more, 2.5×10⁴infectious units or more, 5×10⁴ infectious units or more, or 10⁵infectious units or more in a subject. In certain embodiments, the virusachieves a viral yield of 1 infectious unit or more, 10 infectious unitsor more, 5×10¹ infectious units or more, 10² infectious units or more,5×10² infectious units or more, 10³ infectious units or more, 2.5×10³infectious units or more, 5×10³ infectious units or more, 10⁴ infectiousunits or more, 2.5×10⁴ infectious units or more, 5×10⁴ infectious unitsor more, or 10⁵ infectious units or more in a subject within 4 hours, 6hours, 8 hours, 12 hours, 16 hours, 24 hours, or 48 hours. In certainembodiments, the virus achieves a viral yield of 1 infectious unit ormore, 10 infectious units or more, 10¹ infectious units or more, 5×10¹infectious units or more, 10² infectious units or more, 5×10² infectiousunits or more, 10³ infectious units or more, 2.5×10³ infectious units ormore, 5×10³ infectious units or more, 10⁴ infectious units or more,2.5×10⁴ infectious units or more, 5×10⁴ infectious units or more, or 10⁵infectious units or more in a subject within 48 hours, 72 hours, or 1week. In accordance with these embodiments, the viral yield may bemeasured in the infected tissue or serum. In a specific embodiment, thesubject is immunocompetent. In another embodiment, the subject isimmunocompromised or immunosuppressed. In a specific embodiment, thevirus achieves a yield of less than 10⁴ infectious units. In otherembodiments the virus achieves a yield of 10⁵ or more infectious units.

In some embodiments, the virus achieves a viral titer of 1 infectiousunit per ml or more, 10 infectious units per ml or more, 5×10¹infectious units per ml or more, 10² infectious units per ml or more,5×10² infectious units per ml or more, 10³ infectious units per ml ormore, 2.5×10³ infectious units per ml or more, 5×10³ infectious unitsper ml or more, 10⁴ infectious units per ml or more, 2.5×10⁴ infectiousunits per ml or more, 5×10⁴ infectious units per ml or more, or 10⁵infectious units per ml or more in a subject. In certain embodiments,the virus achieves a viral titer of 10 infectious units per ml or more,5×10¹ infectious units per ml or more, 10² infectious units per ml ormore, 5×10² infectious units per ml or more, 10³ infectious units per mlor more, 2.5×10³ infectious units per ml or more, 5×10³ infectious unitsper ml or more, 10⁴ infectious units per ml or more, 2.5×10⁴ infectiousunits per ml or more, 5×10⁴ infectious units per ml or more, or 10⁵infectious units per ml or more in a subject within 4 hours, 6 hours, 8hours, 12 hours, 16 hours, 24 hours, or 48 hours. In certainembodiments, the virus achieves a viral titer of 1 infectious unit permL or more, 10 infectious units per ml or more, 5×10¹ infectious unitsper ml or more, 10² infectious units per ml or more, 5×10² infectiousunits per ml or more, 10³ infectious units per mL or more, 2.5×10³infectious units per ml or more, 5×10³ infectious units per ml or more,10⁴ infectious units per ml or more, 2.5×10⁴ infectious units per ml ormore, 5×10⁴ infectious units per ml or more, or 10⁵ infectious units perml or more in a subject within 48 hours, 72 hours, or 1 week. Inaccordance with these embodiments, the viral titer may be measured inthe infected tissue or serum. In a specific embodiment, the subject isimmunocompetent. In another embodiment, the subject is immunocompromisedor immunosuppressed. In a specific embodiment, the virus achieves atiter of less than 10⁴ infectious units per ml. In some embodiments, thevirus achieves 10⁵ or more infectious units per ml.

In some embodiments, the virus infects a cell and produces, 10¹ or more,2.5×10¹ or more, 5×10¹ or more, 7.5×10¹ or more, 10² or more, 2.5×10² ormore, 5×10² or more, 7.5×10² or more, 10³ or more, 2.5×10³ or more,5×10³ or more, 7.5×10³ or more, 10⁴ or more, 2.5×10⁴ or more, 5×10⁴ ormore, 7.5×10⁴ or more, or 10⁵ or more viral particles per cell. Incertain embodiments, the virus infects a cell and produces 10 or more,10¹ or more, 2.5×10¹ or more, 5×10¹ or more, 7.5×10¹ or more, 10² ormore, 2.5×10² or more, 5×10² or more, 7.5×10² or more, 10³ or more,2.5×10³ or more, 5×10³ or more, 7.5×10³ or more, 10⁴ or more, 2.5×10⁴ ormore, 5×10⁴ or more, 7.5×10⁴ or more, or 10⁵ or more viral particles percell within 4 hours, 6 hours, 8 hours, 12 hours, 16 hours, or 24 hours.In other embodiments, the virus infects a cell and produces 10 or more,10¹ or more, 2.5×10¹ or more, 5×10¹ or more, 7.5×10¹ or more, 10² ormore, 2.5×10² or more, 5×10² or more, 7.5×10² or more, 10³ or more,2.5×10³ or more, 5×10³ or more, 7.5×10³ or more, 10⁴ or more, 2.5×10⁴ ormore, 5×10⁴ or more, 7.5×10⁴ or more, or 10⁵ or more viral particles percell within 48 hours, 72 hours, or 1 week.

In other embodiments, the virus is latent for a period of about at least1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days,10 days, 11 days, 12 days, 13 days, 14 days, or 15 days. In anotherembodiment, the virus is latent for a period of about at least 1 week,or 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9weeks, or 10 weeks. In a further embodiment, the virus is latent for aperiod of about at least 1 month, 2 months, 3 months, 4 months, 5months, 6 months, 7 months, 8 months, 9 months, 10 months, or 11 months.In yet another embodiment, the virus is latent for a period of about atleast 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, or 15years. In some embodiments, the virus is latent for a period of greaterthan 15 years.

4.2 In vitro Assays to Detect Antiviral Activity

The antiviral activity of compounds may be assessed in various in vitroassays described herein or others known to one of skill in the art.Non-limiting examples of the viruses that can be tested for compoundswith antiviral activities against such viruses are provided in Section4.1, supra. In specific embodiments, compounds exhibit an activityprofile that is consistent with their ability to inhibit viralreplication while maintaining low toxicity with respect to eukaryoticcells, preferably mammalian cells. For example, the effect of a compoundon the replication of a virus may be determined by infecting cells withdifferent dilutions of a virus in the presence or absence of variousdilutions of a compound, and assessing the effect of the compound on,e.g., viral replication, viral genome replication, and/or the synthesisof viral proteins. Alternatively, the effect of a compound on thereplication of a virus may be determined by contacting cells withvarious dilutions of a compound or a placebo, infecting the cells withdifferent dilutions of a virus, and assessing the effect of the compoundon, e.g., viral replication, viral genome replication, and/or thesynthesis of viral proteins. Altered viral replication can be assessedby, e.g., plaque formation. The production of viral proteins can beassessed by, e.g., ELISA, Western blot, immunofluorescence, or flowcytometry analysis. The production of viral nucleic acids can beassessed by, e.g., RT-PCR, PCR, Northern blot analysis, or Southernblot.

In certain embodiments, compounds reduce the replication of a virus byapproximately 10%, preferably 15%, 25%, 30%, 45%, 50%, 60%, 75%, 95% ormore relative to a negative control (e.g., PBS, DMSO) in an assaydescribed herein or others known to one of skill in the art. In someembodiments, compounds reduce the replication of a virus by about atleast 1.5 fold, 2, fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold,9 fold, 10 fold, 15 fold, 20 fold, 25 fold, 30 fold, 35 fold, 40 fold,45 fold, 50 fold, 75 fold, 100 fold, 500 fold, or 1000 fold relative toa negative control (e.g., PBS, DMSO) in an assay described herein orothers known to one of skill in the art. In other embodiments, compoundsreduce the replication of a virus by about at least 1.5 to 3 fold, 2 to4 fold, 3 to 5 fold, 4 to 8 fold, 6 to 9 fold, 8 to 10 fold, 2 to 10fold, 5 to 20 fold, 10 to 40 fold, 10 to 50 fold, 25 to 50 fold, 50 to100 fold, 75 to 100 fold, 100 to 500 fold, 500 to 1000 fold, or 10 to1000 fold relative to a negative control (e.g., PBS, DMSO) in an assaydescribed herein or others known to one of skill in the art. In otherembodiments, compounds reduce the replication of a virus by about 1 log,1.5 logs, 2 logs, 2.5 logs, 3 logs, 3.5 logs, 4 logs, 4.5 logs, 5 logsor more relative to a negative control (e.g., PBS, DMSO) in an assaydescribed herein or others known to one of skill in the art. Inaccordance with these embodiments, such compounds may be furtherassessed for their safety and efficacy in assays such as those describedin Section 4, infra.

In certain embodiments, compounds reduce the replication of a viralgenome by approximately 10%, preferably 15%, 25%, 30%, 45%, 50%, 60%,75%, 95% or more relative to a negative control (e.g., PBS, DMSO) in anassay described herein or others known to one of skill in the art. Insome embodiments, compounds reduce the replication of a viral genome byabout at least 1.5 fold, 2, fold, 3 fold, 4 fold, 5 fold, 6 fold, 7fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 25 fold, 30 fold, 35fold, 40 fold, 45 fold, 50 fold, 75 fold, 100 fold, 500 fold, or 1000fold relative to a negative control (e.g., PBS, DMSO) in an assaydescribed herein or others known to one of skill in the art. In otherembodiments, compounds reduce the replication of a viral genome by aboutat least 1.5 to 3 fold, 2 to 4 fold, 3 to 5 fold, 4 to 8 fold, 6 to 9fold, 8 to 10 fold, 2 to 10 fold, 5 to 20 fold, 10 to 40 fold, 10 to 50fold, 25 to 50 fold, 50 to 100 fold, 75 to 100 fold, 100 to 500 fold,500 to 1000 fold, or 10 to 1000 fold relative to a negative control(e.g., PBS, DMSO) in an assay described herein or others known to one ofskill in the art. In other embodiments, compounds reduce the replicationof a viral genome by about 1 log, 1.5 logs, 2 logs, 2.5 logs, 3 logs,3.5 logs, 4 logs, 4.5 logs, 5 logs or more relative to a negativecontrol (e.g., PBS, DMSO) in an assay described herein or others knownto one of skill in the art. In accordance with these embodiments, suchcompounds may be further assessed for their safety and efficacy inassays such as those described in Section 4, infra.

In certain embodiments, compounds reduce the synthesis of viral proteinsby approximately 10%, preferably 15%, 25%, 30%, 45%, 50%, 60%, 75%, 95%or more relative to a negative control (e.g., PBS, DMSO) in an assaydescribed herein or others known to one of skill in the art. In someembodiments, compounds reduce the synthesis of viral proteins byapproximately at least 1.5 fold, 2, fold, 3 fold, 4 fold, 5 fold, 6fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 25 fold, 30fold, 35 fold, 40 fold, 45 fold, 50 fold, 75 fold, 100 fold, 500 fold,or 1000 fold relative to a negative control (e.g., PBS, DMSO) in anassay described herein or others known to one of skill in the art. Inother embodiments, compounds reduce the synthesis of viral proteins byapproximately at least 1.5 to 3 fold, 2 to 4 fold, 3 to 5 fold, 4 to 8fold, 6 to 9 fold, 8 to 10 fold, 2 to 10 fold, 5 to 20 fold, 10 to 40fold, 10 to 50 fold, 25 to 50 fold, 50 to 100 fold, 75 to 100 fold, 100to 500 fold, 500 to 1000 fold, or 10 to 1000 fold relative to a negativecontrol (e.g., PBS, DMSO) in an assay described herein or others knownto one of skill in the art. In other embodiments, compounds reduce thesynthesis of viral proteins by approximately 1 log, 1.5 logs, 2 logs,2.5 logs, 3 logs, 3.5 logs, 4 logs, 4.5 logs, 5 logs or more relative toa negative control (e.g., PBS, DMSO) in an assay described herein orothers known to one of skill in the art. In accordance with theseembodiments, such compounds may be further assessed for their safety andefficacy in assays such as those described in Section 4.3, infra.

In some embodiments, compounds result in about a 1.5 fold or more, 2fold or more, 3 fold or more, 4 fold or more, 5 fold or more, 6 fold ormore, 7 fold or more, 8 fold or more, 9 fold or more, 10 fold or more,15 fold or more, 20 fold or more, 25 fold or more, 30 fold or more, 35fold or more, 40 fold or more, 45 fold or more, 50 fold or more, 60 foldor more, 70 fold or more, 80 fold or more, 90 fold or more, or 100 foldor more inhibition/reduction of viral yield per round of viralreplication. In certain embodiments, compounds result in about a 2 foldor more reduction inhibition/reduction of viral yield per round of viralreplication. In specific embodiments, compounds result in about a 10fold or more inhibition/reduction of viral yield per round of viralreplication.

The in vitro antiviral assays can be conducted using any eukaryoticcell, including primary cells and established cell lines. The cell orcell lines selected should be susceptible to infection by a virus ofinterest. Non-limiting examples of mammalian cell lines that can be usedin standard in vitro antiviral assays (e.g., viral cytopathic effectassays, neutral red update assays, viral yield assay, plaque reductionassays) for the respective viruses are set out in Table 5.

TABLE 5 Examples of Mammalian Cell Lines in Antiviral Assays Virus CellLine herpes simplex virus (HSV) primary fibroblasts (MRC-5 cells) Verocells human cytomegalovirus primary fibroblasts (MRC-5 cells) (HCMV)Influenza primary fibroblasts (MRC-5 cells) Madin Darby canine kidney(MDCK) primary chick embryo chick kidney calf kidney African greenmonkey kidney (Vero) cells mink lung human respiratory epithelia cellshepatitis C virus Huh7 (or Huh7.7) Huh7.5 primary human hepatocytes(PHH) immortalized human hepatocytes (IHH) HIV-1 MT-2 cells (T cells)Dengue virus Vero cells Measles virus African green monkey kidney (CV-1)cells SARS virus Vero 76 cells Respiratory syncytial virus African greenmonkey kidney (MA-104) cells Venezuelan equine Vero cells encephalitisvirus West Nile virus Vero cells yellow fever virus Vero cells HHV-6Cord Blood Lymphocytes (CBL) Human T cell lymphoblastoid cell lines(HSB-2 and SupT-1) HHV-8 B-cell lymphoma cell line (BCBL-1) EBVumbilical cord blood lymphocytes

Sections 4.2.1 to 4.2.7 below provide non-limiting examples of antiviralassays that can be used to characterize the antiviral activity ofcompounds against the respective virus. One of skill in the art willknow how to adapt the methods described in Sections 4.2.1 to 4.2.7 toother viruses by, e.g., changing the cell system and viral pathogen,such as described in Table 5.

4.2.1 Viral Cytopathic Effect (CPE) Assay

CPE is the morphological changes that cultured cells undergo upon beinginfected by most viruses. These morphological changes can be observedeasily in unfixed, unstained cells by microscopy. Forms of CPE, whichcan vary depending on the virus, include, but are not limited to,rounding of the cells, appearance of inclusion bodies in the nucleusand/or cytoplasm of infected cells, and formation of syncytia, orpolykaryocytes (large cytoplasmic masses that contain many nuclei). Foradenovirus infection, crystalline arrays of adenovirus capsidsaccumulate in the nucleus to form an inclusion body.

The CPE assay can provide a measure of the antiviral effect of acompound. In a non-limiting example of such an assay, compounds areserially diluted (e.g. 1000, 500, 100, 50, 10, 1 μg/ml) and added to 3wells containing a cell monolayer (preferably mammalian cells at 80-100%confluent) of a 96-well plate. Within 5 minutes, viruses are added andthe plate sealed, incubated at 37° C. for the standard time periodrequired to induce near-maximal viral CPE (e.g., approximately 48 to 120hours, depending on the virus and multiplicity of infection). CPE isread microscopically after a known positive control drug is evaluated inparallel with compounds in each test. Non-limiting examples of positivescontrols are ribavirin for dengue, influenza, measles, respiratorysyncytial, parainfluenza, Pichinde, Punta Toro and Venezuelan equineencephalitis viruses; cidofovir for adenovirus; pirodovir forrhinovirus; 6-azauridine for West Nile and yellow fever viruses; andalferon (interferon α-n3) for SARS virus. The data are expressed as 50%effective concentrations or approximated virus-inhibitory concentration,50% endpoint (EC50) and cell-inhibitory concentration, 50% endpoint(IC50). General selectivity index (“SI”) is calculated as the IC50divided by the EC50. These values can be calculated using any methodknown in the art, e.g., the computer software program MacSynergy II byM. N. Prichard, K. R. Asaltine, and C. Shipman, Jr., University ofMichigan, Ann Arbor, Mich.

In one embodiment, a compound has an SI of greater than 3, or 4, or 5,or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 20,or 21, or 22, or 23, or 24, or 25, or 30, or 35, or 40, or 45, or 50, or60, or 70, or 80, or 90, or 100, or 200, or 300, or 400, or 500, 1,000,or 10,000. In some embodiments, a compound has an SI of greater than 10.In a specific embodiment, compounds with an SI of greater than 10 arefurther assessed in other in vitro and in vivo assays described hereinor others known in the art to characterize safety and efficacy.

4.2.2 Neutral Red (NR) Dye Uptake Assay

The NR Dye Uptake assay can be used to validate the CPE inhibition assay(See Section 4.2.1). In a non-limiting example of such an assay, thesame 96-well microplates used for the CPE inhibition assay can be used.Neutral red is added to the medium, and cells not damaged by virus takeup a greater amount of dye. The percentage of uptake indicating viablecells is read on a microplate autoreader at dual wavelengths of 405 and540 nm, with the difference taken to eliminate background. (See McManuset al., Appl. Environment. Microbiol. 31:35-38, 1976). An EC50 isdetermined for samples with infected cells and contacted with compounds,and an IC50 is determined for samples with uninfected cells contactedwith compounds.

4.2.3 Virus Yield Assay

Lysed cells and supernatants from infected cultures such as those in theCPE inhibition assay (See section 4.2.1) can be used to assay for virusyield (production of viral particles after the primary infection). In anon-limiting example, these supernatants are serial diluted and addedonto monolayers of susceptible cells (e.g., Vero cells). Development ofCPE in these cells is an indication of the presence of infectiousviruses in the supernatant. The 90% effective concentration (EC90), thetest compound concentration that inhibits virus yield by 1 log₁₀, isdetermined from these data using known calculation methods in the art.In one embodiment, the EC90 of compound is at least 1.5 fold, 2 fold, 3fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 20 fold,30 fold, 40 fold, or 50 fold less than the EC90 of the negative controlsample.

4.2.4 Plaque Reduction Assay

In a non-limiting example of such an assay, the virus is diluted intovarious concentrations and added to each well containing a monolayer ofthe target mammalian cells in triplicate. The plates are then incubatedfor a period of time to achieve effective infection of the controlsample (e.g., 1 hour with shaking every fifteen minutes). After theincubation period, an equal amount of 1% agarose is added to an equalvolume of each compound dilution prepared in 2× concentration. Incertain embodiments, final compound concentrations between 0.03 μg/ml to100 μg/ml can be tested with a final agarose overlay concentration of0.5%. The drug agarose mixture is applied to each well in 2 ml volumeand the plates are incubated for three days, after which the cells arestained with a 1.5% solution of neutral red. At the end of the 4-6 hourincubation period, the neutral red solution is aspirated, and plaquescounted using a stereomicroscope. Alternatively, a final agaroseconcentration of 0.4% can be used. In other embodiments, the plates areincubated for more than three days with additional overlays beingapplied on day four and on day 8 when appropriate. In anotherembodiment, the overlay medium is liquid rather than semi-solid.

4.2.5 Virus Titer Assay

In this non-limiting example, a monolayer of the target mammalian cellline is infected with different amounts (e.g., multiplicity of 3 plaqueforming units (pfu) or 5 pfu) of virus (e.g., HCMV or HSV) andsubsequently cultured in the presence or absence of various dilutions ofcompounds (e.g., 0.1 μg/ml, 1 μg/ml, 5 μg/ml, or 10 μg/ml). Infectedcultures are harvested 48 hours or 72 hours post infection and titeredby standard plaque assays known in the art on the appropriate targetcell line (e.g., Vero cells, MRC5 cells). In certain embodiments,culturing the infected cells in the presence of compounds reduces theyield of infectious virus by at least 1.5 fold, 2, fold, 3, fold, 4fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold,25 fold, 30 fold, 35 fold, 40 fold, 45 fold, 50 fold, 100 fold, 500fold, or 1000 fold relative to culturing the infected cells in theabsence of compounds. In a specific embodiment, culturing the infectedcells in the presence of compounds reduces the PFU/ml by at least 10fold relative to culturing the infected cells in the absence ofcompounds.

In certain embodiments, culturing the infected cells in the presence ofcompounds reduces the yield of infectious virus by at least 0.5 log 10,1 log 10, 1.5 log 10, 2 log 10, 2.5 log 10, 3 log 10, 3.5 log 10, 4 log10, 4.5 log 10, 5 log 10, 5.5 log 10, 6 log 10, 6.5 log 10, 7 log 10,7.5 log 10, 8 log 10, 8.5 log 10, or 9 log 10 relative to culturing theinfected cells in the absence of compounds. In a specific embodiment,culturing the infected cells in the presence of compounds reduces theyield of infectious virus by at least 1 log 10 or 2 log 10 relative toculturing the infected cells in the absence of compounds. In anotherspecific embodiment, culturing the infected cells in the presence ofcompounds reduces the yield of infectious virus by at least 2 log 10relative to culturing the infected cells in the absence of compounds.

4.2.6 Flow Cytometry Assay

Flow cytometry can be utilized to detect expression of virus antigens ininfected target cells cultured in the presence or absence of compounds(See, e.g., McSharry et al., Clinical Microbiology Rev., 1994,7:576-604). Non-limiting examples of viral antigens that can be detectedon cell surfaces by flow cytometry include, but are not limited to gB,gC, gC, and gE of HSV; E protein of Japanese encephalitis; virus gp52 ofmouse mammary tumor virus; gpI of varicella-zoster virus; gB of HCMV;gp160/120 of HIV; HA of influenza; gp110/60 of HHV-6; and H and F ofmeasles virus. In other embodiments, intracellular viral antigens orviral nucleic acid can be detected by flow cytometry with techniquesknown in the art.

4.2.7 Genetically Engineered Cell Lines for Antiviral Assays

Various cell lines for use in antiviral assays can be geneticallyengineered to render them more suitable hosts for viral infection orviral replication and more convenient substrates for rapidly detectingvirus-infected cells (See, e.g., Olivo, P. D., Clin. Microbiol. Rev.,1996, 9:321-334). In some aspects, these cell lines are available fortesting the antiviral activity of compound on blocking any step of viralreplication, such as, transcription, translation, pregenomeencapsidation, reverse transcription, particle assembly and release.Nonlimiting examples of genetically engineered cells lines for use inantiviral assays with the respective virus are discussed below.

HepG2-2.2.15 is a stable cell line containing the hepatitis B virus(HBV) ayw strain genome that is useful in identifying and characterizingcompounds blocking any step of viral replication, such as,transcription, translation, pregenome encapsidation, reversetranscription, particle assembly and release. In one aspect, compoundscan be added to HepG2-2.2.15 culture to test whether compound willreduce the production of secreted HBV from cells utilizing real timequantitative PCR (TaqMan) assay to measure HBV DNA copies. Specifically,confluent cultures of HepG2-2.2.15 cells cultured on 96-wellflat-bottomed tissue culture plates and are treated with variousconcentration of daily doses of compounds. HBV virion DNA in the culturemedium can be assessed 24 hours after the last treatment by quantitativeblot hybridization or real time quantitative PCR (TaqMan) assay. Uptakeof neutral red dye (absorbance of internalized dye at 510 nM [A510]) canbe used to determine the relative level of toxicity 24 hours followingthe last treatment. Values are presented as a percentage of the averageA510 values for separate cultures of untreated cells maintained on thesame plate. Intracellular HBV DNA replication intermediates can beassessed by quantitative Southern blot hybridization. Intracellular HBVparticles can be isolated from the treated HepG2-2.2.15 cells and thepregenomic RNA examined by Southern blot analysis. ELISAs can be used toquantify the amounts of the HBV envelope protein, surface antigen(HBsAg), and secreted e-antigen (HBeAg) released from cultures.Lamivudine (3TC) can be used as a positive assay control. (See Korba &Gerin, Antivir. Res. 19:55-70, 1992).

In one aspect, the cell line Huh7 ET (luc-ubi-neo/ET), which contains anew HCV RNA replicon with a stable luciferase (LUC) reporter, can beused to assay compounds antiviral activity against hepatitis C viralreplication (See Krieger, N., V. Lohmann, and R. Bartenschlager J.Virol., 2001, 75:4614-4624). The activity of the LUC reporter isdirectly proportional to HCV RNA levels and positive control antiviralcompounds behave comparably using either LUC or RNA endpoints.Subconfluent cultures of Huh7 ET cells are plated onto 96-well plates,compounds are added to the appropriate wells the next day, and thesamples as well as the positive (e.g., human interferon-alpha 2b) andnegative control samples are processed 72 hr later when the cells arestill subconfluent. The HCV RNA levels can also be assessed usingquantitative PCR (TaqMan). In some embodiments, compounds reduce the LUCsignal (or HCV RNA levels) by 20%, 35%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 90%, or 95% or more relative to the untreatedsample controls. In a preferred embodiment, compounds reduce the LUCsignal (or HCV RNA levels) by 50% or more relative to the untreated cellcontrols. Other relevant cell culture models to study HCV have beendescribed, e.g., See Durantel et al., J. Hepatology, 2007, 46:1-5.

The antiviral effect of compound can be assayed against EBV by measuringthe level of viral capsid antigen (VCA) production in Daudi cells usingan ELISA assay. Various concentrations of compounds are tested (e.g., 50mg/ml to 0.03 mg/ml), and the results obtained from untreated andcompound treated cells are used to calculate an EC50 value. Selectedcompounds that have good activity against EBV VCA production withouttoxicity will be tested for their ability to inhibit EBV DNA synthesis.

For assays with HSV, the BHKICP6LacZ cell line, which was stablytransformed with the E. coli lacZ gene under the transcriptional controlof the HSV-1 UL39 promoter, can be used (See Stabell et al., 1992,Methods 38:195-204). Infected cells are detected using β-galactosidaseassays known in the art, e.g., colorimetric assay.

Standard antiviral assays for influenza virus has been described, See,e.g., Sidwell et al., Antiviral Research, 2000, 48:1-16. These assayscan also be adapted for use with other viruses.

4.2.8 Approach To Identifying and Measuring Metabolic Fluxes Regulatedby Viral Infection and Anti-Viral Compounds

Viruses can alter cellular metabolic activity through a variety ofroutes. These include affecting transcription, translation, and/ordegradation of mRNAs and/or proteins, relocalization of mRNAs and/orproteins, covalent modification of proteins, and allosteric regulationof enzymes or other proteins; and alterations to the composition ofprotein-containing complexes that modify their activity. The net resultof all of these changes is modulation of metabolic fluxes to meet theneeds of the virus. Thus, metabolic flux changes represent the ultimateendpoint of the virus' efforts to modulate host cell metabolism.Accordingly, fluxes that are increased by the virus are especiallylikely to be critical to viral survival and replication and to representvaluable drug targets.

A novel approach has been developed to profile metabolic fluxes. Itbuilds upon an approach to measuring nitrogen metabolic fluxes in E.coli developed by Rabinowitz and colleagues (Yuan et al., 2006, Nat.Chem. Biol. 2:529-530), which is incorporated herein by reference. Theessence of this kinetic flux profiling (KFP) approach is as follows:

(1) Cells (either uninfected or infected with virus) are rapidlyswitched from unlabeled to isotope-labeled nutrient (or vice versa); forthe present purposes, preferred nutrients include uniformly or partially¹³C-labeled or ¹⁵N-labeled glucose, glutamine, glutamate, or relatedcompounds including without limitation pyruvate, lactate, glycerol,acetate, aspartate, arginine, and urea. Labels can include all knownisotopes of H, C, N, O, P, or S, including both stable and radioactivelabels. Results are dependent on the interplay between the host celltype and the viral pathogen, including the viral load and time postinfection.

(2) Metabolism is quenched at various time points following theisotope-switch (e.g., 0.2, 0.5, 1, 2, 5, 10, 20, 30 min and 1, 2, 4, 8,12, 16, 24, 36, 48 h or a subset or variant thereof). One convenientmeans of metabolism quenching is addition of cold (e.g., dry-icetemperature) methanol, although other solvents and temperatures,including also boiling solvents, are possible.

(3) The metabolome, including its extent of isotope labeling, isquantified for each collected sample. One convenient means of suchquantitation is extraction of metabolites from the cells followed byliquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis ofthe extract. Appropriate extraction protocols and LC-MS/MS methods areknown in the art. See the following citations, which are hereinincorporated by reference (Bajad et al., 2006, J Chromatogr. A1125:76-88; Bolling and Fiehn, 2005, Plant Physiol. 139:1995-2005;Coulier et al., 2006, Anal Chem. 78:6573-6582; Kimball and Rabinowitz,2006, Anal Biochem. 358:273-280; Lu et al., 2006, J. Am. Soc. MassSpectrom. 17:37-50; Lu et al., 2007, J Am Soc Mass Spectrom. 18:898-909;Luo et al., 2007, J. Chromatogr. A 1147:153-164; Maharjan and Ferenci,2003, Anal Biochem 313:145-154; Milne et al., 2006, Methods 39:92-103;Munger et al., 2006, PLoS Pathog. 2:e132; Olsson et al., 2004, AnalChem. 76:2453-2461; Rabinowitz and Kimball, 2007, Anal Chem. 79:6167-73;Schaub et al., 2006, Biotechnol. Prog. 22:1434-1442; van Winden et al.,2005, FEMS Yeast Research 5:559-568; Villas-Boas et al., 2005, Yeast22:1155-1169.; Wittmann et al., 2004, Anal Biochem. 327:135-139; Wu etal., 2005, Anal Biochem. 336:164-171; Yuan et al., 2006, Nat. Chem.Biol. 2:529-530).

(4) The resulting data is analyzed to determine the cellular metabolicfluxes.

The KFP data is analyzed based on the following principles, throughwhose application those skilled in the art of cellular metabolism canidentify flux changes associated with viral infection by comparingresults for infected versus uninfected samples:

(1) Metabolites closer to the added nutrient in the metabolic networkwill become labeled before their downstream products. Thus, the patternof labeling provides insight into the route taken to forming aparticular metabolite. For example, more rapid labeling of oxaloacetatethan citrate upon switching cells from unlabeled to uniformly¹³C-labeled glucose would imply formation of oxaloacetate viaphosphoenolpyruvate carboxylase or phosphoenolpyruvate carboxykinaserather than via clockwise turning of the tricarboxylic acid cycle.

(2) The speed of labeling provides insight into the quantitative fluxthrough different metabolic pathways, with fast labeling of a metabolitepool resulting from large flux through that pool and/or low absolutepool size of it. For the ideal case of a well-mixed system in which anutrient is being directly converted into an intracellular metabolite,instantaneous switching of the nutrient input into isotope-labeled form,without other modulation of the system, results over time indisappearance of the unlabeled metabolite:

dX ^(U) /dt=−f _(X) X ^(U) /X ^(T)  Eq. (A)

where X^(T) is the total pool of metabolite X; X^(U) the unlabeled form;and f_(X) is the sum of all fluxes consuming the metabolite. For f_(X)and X^(T) constant (i.e., the system at pseudo-steady-state prior to theisotope switch),

X ^(U) /X ^(T)=exp(−f _(X) t/X ^(T))  Eq. (B)

and f _(X) =X ^(T) k _(X)  Eq. (C)

where k_(X) is the apparent first-order rate constant for disappearanceof the unlabeled metabolite. According to Eq. (C), the total fluxthrough metabolite X can be determined based on two parameters that canbe measured directly experimentally: the intracellular pool size of themetabolite and the rate of disappearance of the unlabeled form. While inpractice isotope switching is not instantaneous and slightly morecomplex equations are required, the full differential equations canstill often be solved analytically and typically involve only two freeparameters, with one of these, k_(X), directly yielding total metabolicflux as shown above (Yuan et al., 2006, Nat. Chem. Biol. 2:529-530).

In certain cases involving branched and cyclic pathways, however, themathematics become more complex and use of more sophisticatedcomputational algorithms to facilitate data analysis may be beneficial.The cellular metabolic network can be described by a system ofdifferential equations describing changes in metabolite levels over time(including changes in isotopic labeling patterns). See the followingcitations, which are hereby incorporated by reference (Reed et al.,2003, Genome Biol. 4:R54; Sauer, 2006, Mol. Syst. Biol. 2:62;Stephanopoulos, 1999, Metab. Eng. 1:1-11; Szyperski et al., 1999, Metab.Eng. 1:189-197; Zupke et al., 1995). Such descriptions, wherein the formof the equations is parallel to Eq. (A) above, can be solved for fluxesf_(x1), f_(x2), etc. based on experimentally observed data describingmetabolite concentrations and labeling kinetics (X^(T) atpseudo-steady-state and X^(U)/X^(T) as a function of time). Oneappropriate class of algorithm for obtaining such solutions is describedin the following citations, which are hereby incorporated by reference(Feng and Rabitz, 2004, Biophys. J. 86:1270-1281; Feng et al., 2006, J.Phys. Chem. A. Mol. Spectrosc. Kinet. Environ. Gen. Theory110:7755-7762).

In general, changes in fluxes induced by viral infections occur slowlyrelative to the turnover of metabolites. Accordingly, the steady-stateassumption generally applies to virally perturbed metabolic networksover short to moderate timescales (e.g., for CMV, up to ˜2 h; the exactlength of time depends on the nature of the viral pathogen, with moreaggressive pathogens generally associated with shorter time scales).

At steady-state, the flux through all steps of a linear metabolicpathway must be equal. Accordingly, if flux through one step of apathway is markedly increased by viral infection, the flux through theother steps is likely also increased. A complication arises due tobranching, however. While the effect of branching is small in the casethat the side branches are associated with low relative flux, thepossibility of branching (as well as non-steady-state conditions) pointsto the need for more experimental data than just one measured pathwayflux to implicate other pathway steps as viable drug targets. Ifincreased flux is experimentally demonstrated at both steps upstream anddownstream of an unmeasured step of the pathway, however, then one canhave greatly increased confidence that the flux at the (unmeasured)intermediate step is also increased. Accordingly, herein we considerdemonstration of increased flux at both the upstream and downstreamsteps (but, in selected embodiments, neither individually) to beadequate to validate the intermediate flux (and associated catalyzingenzyme) as a valid antiviral drug target.

4.3 Characterization of Safety and Efficacy of Compounds

The safety and efficacy of compounds can be assessed using technologiesknown to one of skill in the art. Sections 4.4 and 4.5 below providenon-limiting examples of cytotoxicity assays and animal model assays,respectively, to characterize the safety and efficacy of compounds. Incertain embodiments, the cytotoxicity assays described in Section 4.4are conducted following the in vitro antiviral assays described inSection 4, supra. In other embodiments, the cytotoxicity assaysdescribed in Section 4.4 are conducted before or concurrently with thein vitro antiviral assays described in Section 4, supra.

In some embodiments, compounds differentially affect the viability ofuninfected cells and cells infected with virus. The differential effectof a compound on the viability of virally infected and uninfected cellsmay be assessed using techniques such as those described in Section 4.4,infra, or other techniques known to one of skill in the art. In certainembodiments, compounds are more toxic to cells infected with a virusthan uninfected cells. In specific embodiments, compounds preferentiallyaffect the viability of cells infected with a virus. Without being boundby any particular concept, the differential effect of a compound on theviability of uninfected and virally infected cells may be the result ofthe compound targeting a particular enzyme or protein that isdifferentially expressed or regulated or that has differentialactivities in uninfected and virally infected cells. For example, viralinfection and/or viral replication in an infected host cells may alterthe expression, regulation, and/or activities of enzymes and/orproteins. Accordingly, in some embodiments, other compounds that targetthe same enzyme, protein or metabolic pathway are examined for antiviralactivity. In other embodiments, congeners of compounds thatdifferentially affect the viability of cells infected with virus aredesigned and examined for antiviral activity. Non-limiting examples ofantiviral assays that can be used to assess the antiviral activity ofcompound are provided in Section 4, supra.

4.4 Cytotoxicity Studies

In a preferred embodiment, the cells are animal cells, including primarycells and cell lines. In some embodiments, the cells are human cells. Incertain embodiments, cytotoxicity is assessed in one or more of thefollowing cell lines: U937, a human monocyte cell line; primaryperipheral blood mononuclear cells (PBMC); Huh7, a human hepatoblastomacell line; 293T, a human embryonic kidney cell line; and THP-1,monocytic cells. Other non-limiting examples of cell lines that can beused to test the cytotoxicity of compounds are provided in Table 5.

Many assays well-known in the art can be used to assess viability ofcells (infected or uninfected) or cell lines following exposure to acompound and, thus, determine the cytotoxicity of the compound. Forexample, cell proliferation can be assayed by measuringBromodeoxyuridine (BrdU) incorporation (See, e.g., Hoshino et al., 1986,Int. J. Cancer 38, 369; Campana et al., 1988, J. Immunol. Meth. 107:79),(3H) thymidine incorporation (See, e.g., Chen, J., 1996, Oncogene13:1395-403; Jeoung, J., 1995, J. Biol. Chem. 270:18367 73), by directcell count, or by detecting changes in transcription, translation oractivity of known genes such as proto-oncogenes (e.g., fos, myc) or cellcycle markers (Rb, cdc2, cyclin A, D1, D2, D3, E, etc). The levels ofsuch protein and mRNA and activity can be determined by any method wellknown in the art. For example, protein can be quantitated by knownimmunodiagnostic methods such as ELISA, Western blotting orimmunoprecipitation using antibodies, including commercially availableantibodies. mRNA can be quantitated using methods that are well knownand routine in the art, for example, using northern analysis, RNaseprotection, or polymerase chain reaction in connection with reversetranscription. Cell viability can be assessed by using trypan-bluestaining or other cell death or viability markers known in the art. In aspecific embodiment, the level of cellular ATP is measured to determinedcell viability.

In specific embodiments, cell viability is measured in three-day andseven-day periods using an assay standard in the art, such as theCellTiter-Glo Assay Kit (Promega) which measures levels of intracellularATP. A reduction in cellular ATP is indicative of a cytotoxic effect. Inanother specific embodiment, cell viability can be measured in theneutral red uptake assay. In other embodiments, visual observation formorphological changes may include enlargement, granularity, cells withragged edges, a filmy appearance, rounding, detachment from the surfaceof the well, or other changes. These changes are given a designation ofT (100% toxic), PVH (partially toxic-very heavy-80%), PH (partiallytoxic-heavy-60%), P (partially toxic-40%), Ps (partiallytoxic-slight-20%), or 0 (no toxicity-0%), conforming to the degree ofcytotoxicity seen. A 50% cell inhibitory (cytotoxic) concentration(IC50) is determined by regression analysis of these data.

Compounds can be tested for in vivo toxicity in animal models. Forexample, animal models, described herein and/or others known in the art,used to test the antiviral activities of compounds can also be used todetermine the in vivo toxicity of these compounds. For example, animalsare administered a range of concentrations of compounds. Subsequently,the animals are monitored over time for lethality, weight loss orfailure to gain weight, and/or levels of serum markers that may beindicative of tissue damage (e.g., creatine phosphokinase level as anindicator of general tissue damage, level of glutamic oxalic acidtransaminase or pyruvic acid transaminase as indicators for possibleliver damage). These in vivo assays may also be adapted to test thetoxicity of various administration mode and/or regimen in addition todosages.

The toxicity and/or efficacy of a compound in accordance with theinvention can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., for determining the LD50(the dose lethal to 50% of the population) and the ED50 (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio LD50/ED50. A compound identified inaccordance with the invention that exhibits large therapeutic indices ispreferred. While a compound identified in accordance with the inventionthat exhibits toxic side effects may be used, care should be taken todesign a delivery system that targets such agents to the site ofaffected tissue in order to minimize potential damage to uninfectedcells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage of a compound identified inaccordance with the invention for use in humans. The dosage of suchagents lies preferably within a range of circulating concentrations thatinclude the ED50 with little or no toxicity. The dosage may vary withinthis range depending upon the dosage form employed and the route ofadministration utilized. For any agent used in the method of theinvention, the therapeutically effective dose can be estimated initiallyfrom cell culture assays. A dose may be formulated in animal models toachieve a circulating plasma concentration range that includes the IC50(i.e., the concentration of the test compound that achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma may be measured, for example, byhigh-performance liquid chromatography. Additional informationconcerning dosage determination is provided in Section 6.4, infra.

4.5 Animal Models

Compounds and compositions are preferably assayed in vivo for thedesired therapeutic or prophylactic activity prior to use in humans. Forexample, in vivo assays can be used to determine whether it ispreferable to administer a compound and/or another therapeutic agent.For example, to assess the use of a compound to prevent a viralinfection, the compound can be administered before the animal isinfected with the virus. In another embodiment, a compound can beadministered to the animal at the same time that the animal is infectedwith the virus. To assess the use of a compound to treat or manage aviral infection, in one embodiment, the compound is administered after aviral infection in the animal. In another embodiment, a compound isadministered to the animal at the same time that the animal is infectedwith the virus to treat and/or manage the viral infection. In a specificembodiment, the compound is administered to the animal more than onetime.

Compounds can be tested for antiviral activity against virus in animalmodels systems including, but are not limited to, rats, mice, chicken,cows, monkeys, pigs, goats, sheep, dogs, rabbits, guinea pigs, etc. In aspecific embodiment of the invention, compounds are tested in a mousemodel system. Such model systems are widely used and well-known to theskilled artisan.

Animals are infected with virus and concurrently or subsequently treatedwith a compound or placebo. Samples obtained from these animals (e.g.,serum, urine, sputum, semen, saliva, plasma, or tissue sample) can betested for viral replication via well known methods in the art, e.g.,those that measure altered viral replication (as determined, e.g., byplaque formation) or the production of viral proteins (as determined,e.g., by Western blot, ELISA, or flow cytometry analysis) or viralnucleic acids (as determined, e.g., by RT-PCR, northern blot analysis orsouthern blot). For quantitation of virus in tissue samples, tissuesamples are homogenized in phosphate-buffered saline (PBS), anddilutions of clarified homogenates are adsorbed for 1 hour at 37° C.onto monolayers of cells (e.g., Vero, CEF or MDCK cells). In otherassays, histopathologic evaluations are performed after infection,preferably evaluations of the organ(s) the virus is known to target forinfection. Virus immunohistochemistry can be performed using aviral-specific monoclonal antibody. Non-limiting exemplary animal modelsdescribed below (Sections 4.5.1-4.5.5) can be adapted for other viralsystems.

The effect of a compound on the virulence of a virus can also bedetermined using in vivo assays in which the titer of the virus in aninfected subject administered a compound, the length of survival of aninfected subject administered a compound, the immune response in aninfected subject administered a compound, the number, duration and/orseverity of the symptoms in an infected subject administered a compound,and/or the time period before onset of one or more symptoms in aninfected subject administered a compound is assessed. Techniques knownto one of skill in the art can be used to measure such effects.

4.5.1 Herpes Simplex Virus (HSV)

Mouse models of herpes simplex virus type 1 or type 2 (HSV-1 or HSV-2)can be employed to assess the antiviral activity of compounds in vivo.BALB/c mice are commonly used, but other suitable mouse strains that aresusceptible can also be used. Mice are inoculated by various routes withan appropriate multiplicity of infection of HSV (e.g., 10⁵ pfu of HSV-1strain E-377 or 4×10⁴ pfu of HSV-2 strain MS) followed by administrationof compounds and placebo. For i.p. inoculation, HSV-1 replicates in thegut, liver, and spleen and spreads to the CNS. For i.n. inoculation,HSV-1 replicates in the nasaopharynx and spreads to the CNS. Anyappropriate route of administration (e.g., oral, topical, systemic,nasal), frequency and dose of administration can be tested to determinethe optimal dosages and treatment regimens using compounds, optionallyin combination with other therapies.

In a mouse model of HSV-2 genital disease, intravaginal inoculation offemale Swiss Webster mice with HSV-1 or HSV-2 is carried out, andvaginal swabs are obtained to evaluate the effect of therapy on viralreplication (See, e.g., Crute et al., Nature Medicine, 2002, 8:386-391).For example, viral titers by plaque assays are determined from thevaginal swabs. A mouse model of HSV-1 using SKH-1 mice, a strain ofimmunocompetent hairless mice, to study cutaneous lesions is alsodescribed in the art (See, e.g., Crute et al., Nature Medicine, 2002,8:386-391 and Bolger et al., Antiviral Res., 1997, 35:157-165). Guineapig models of HSV have also been described, See, e.g., Chen et al.,Virol. J, 2004 Nov. 23, 1:11. Statistical analysis is carried out tocalculate significance (e.g., a P value of 0.05 or less).

4.5.2 HCMV

Since HCMV does not generally infect laboratory animals, mouse models ofinfection with murine CMV (MCMV) can be used to assay antiviral activitycompounds in vivo. For example, a MCMV mouse model with BALB/c mice canbe used to assay the antiviral activities of compounds in vivo whenadministered to infected mice (See, e.g., Kern et al., Antimicrob.Agents Chemother., 2004, 48:4745-4753). Tissue homogenates isolated frominfected mice treated or untreated with compounds are tested usingstandard plaque assays with mouse embryonic fibroblasts (MEFs).Statistical analysis is then carried out to calculate significance(e.g., a P value of 0.05 or less).

Alternatively, human tissue (i.e., retinal tissue or fetal thymus andliver tissue) is implanted into SCID mice, and the mice are subsequentlyinfected with HCMV, preferably at the site of the tissue graft (See,e.g., Kern et al., Antimicrob. Agents Chemother., 2004, 48:4745-4753).The pfu of HCMV used for inoculation can vary depending on theexperiment and virus strain. Any appropriate routes of administration(e.g., oral, topical, systemic, nasal), frequency and dose ofadministration can be tested to determine the optimal dosages andtreatment regimens using compounds, optionally in combination with othertherapies. Implant tissue homogenates isolated from infected micetreated or untreated with compounds at various time points are testedusing standard plaque assays with human foreskin fibroblasts (HFFs).Statistical analysis is then carried out to calculate significance(i.e., a P value of 0.05 or less).

Guinea pig models of CMV to study antiviral agents have also beendescribed, See, e.g., Bourne et al., Antiviral Res., 2000, 47:103-109;Bravo et al., Antiviral Res., 2003, 60:41-49; and Bravo et al, J.Infectious Diseases, 2006, 193:591-597.

4.5.3 Influenza

Animal models, such as ferret, mouse and chicken, developed for use totest antiviral agents against influenza virus have been described, See,e.g., Sidwell et al., Antiviral Res., 2000, 48:1-16; and McCauley etal., Antiviral Res., 1995, 27:179-186. For mouse models of influenza,non-limiting examples of parameters that can be used to assay antiviralactivity of compounds administered to the influenza-infected miceinclude pneumonia-associated death, serum al-acid glycoprotein increase,animal weight, lung virus assayed by hemagglutinin, lung virus assayedby plaque assays, and histopathological change in the lung. Statisticalanalysis is carried out to calculate significance (e.g., a P value of0.05 or less).

Nasal turbinates and trachea may be examined for epithelial changes andsubepithelial inflammation. The lungs may be examined for bronchiolarepithelial changes and peribronchiolar inflammation in large, medium,and small or terminal bronchioles. The alveoli are also evaluated forinflammatory changes. The medium bronchioles are graded on a scale of 0to 3+ as follows: 0 (normal: lined by medium to tall columnar epithelialcells with ciliated apical borders and basal pseudostratified nuclei;minimal inflammation); 1+ (epithelial layer columnar and even in outlinewith only slightly increased proliferation; cilia still visible on manycells); 2+ (prominent changes in the epithelial layer ranging fromattenuation to marked proliferation; cells disorganized and layeroutline irregular at the luminal border); 3+(epithelial layer markedlydisrupted and disorganized with necrotic cells visible in the lumen;some bronchioles attenuated and others in marked reactiveproliferation).

The trachea is graded on a scale of 0 to 2.5+ as follows: 0 (normal:Lined by medium to tall columnar epithelial cells with ciliated apicalborder, nuclei basal and pseudostratified. Cytoplasm evident betweenapical border and nucleus. Occasional small focus with squamous cells);1+ (focal squamous metaplasia of the epithelial layer); 2+ (diffusesquamous metaplasia of much of the epithelial layer, cilia may beevident focally); 2.5+ (diffuse squamous metaplasia with very few ciliaevident).

Virus immunohistochemistry is performed using a viral-specificmonoclonal antibody (e.g. NP-, N- or HN-sepcific monoclonal antibodies).Staining is graded 0 to 3+ as follows: 0 (no infected cells); 0.5+ (fewinfected cells); 1+ (few infected cells, as widely separated individualcells); 1.5+ (few infected cells, as widely separated singles and insmall clusters); 2+ (moderate numbers of infected cells, usuallyaffecting clusters of adjacent cells in portions of the epithelial layerlining bronchioles, or in small sublobular foci in alveoli); 3+(numerous infected cells, affecting most of the epithelial layer inbronchioles, or widespread in large sublobular foci in alveoli).

4.5.4 Hepatitis

A HBV transgenic mouse model, lineage 1.3.46 (official designation,Tg[HBV 1.3 genome] Chi46) has been described previously and can be usedto test the in vivo antiviral activities of compounds as well as thedosing and administration regimen (See, e.g., Cavanaugh et al., J.Virol., 1997, 71:3236-3243; and Guidotti et al., J. Virol., 1995,69:6158-6169). In these HBV transgenic mice, a high level of viralreplication occurs in liver parenchymal cells and in the proximalconvoluted tubules in the kidneys of these transgenic mice at levelscomparable to those observed in the infected liver of patients withchronic HBV hepatitis. HBV transgenic mice that have been matched forage (i.e., 6-10 weeks), sex (i.e., male), and levels of hepatitis Bsurface antigen (HBsAg) in serum can be treated with compounds orplacebo followed by antiviral activity analysis to assess the antiviralactivity of compounds. Non-limiting examples of assays that can beperformed on these mice treated and untreated with compounds includeSouthern analysis to measure HBV DNA in the liver, quantitative reversetranscriptase PCR (qRT-PCR) to measure HBV RNA in liver, immunoassays tomeasure hepatitis e antigen (HBeAg) and HBV surface antigen (HBsAg) inthe serum, immunohistochemistry to measure HBV antigens in the liver,and quantitative PCR (qPCR) to measure serum HBV DNA. Gross andmicroscopic pathological examinations can be performed as needed.

Various hepatitis C virus (HCV) mouse models described in the art can beused in assessing the antiviral activities of compounds against HCVinfection (See Zhu et al., Antimicrobial Agents and Chemother., 2006,50:3260-3268; Bright et al., Nature, 2005, 436:973-978; Hsu et al., Nat.Biotechnol., 2003, 21:519-525; Ilan et al., J. Infect. Dis. 2002,185:153-161; Kneteman et al., Hepatology, 2006, 43:1346-1353; Mercer etal., Nat. Med., 2001, 7:927-933; and Wu et al., Gastroenterology, 2005,128:1416-1423). For example, mice with chimeric human livers aregenerated by transplanting normal human hepatocytes into SCID micecarrying a plasminogen activator transgene (Alb-uPA) (See Mercer et al.,Nat. Med., 2001, 7:927-933). These mice can develop prolonged HCVinfections with high viral titers after inoculation with HCV (e.g., frominfected human serum). Thus, these mice can be administered a compoundor placebo prior to, concurrently with, or subsequent to HCV infection,and replication of the virus can be confirmed by detection ofnegative-strand viral RNA in transplanted livers or expression of HCVviral proteins in the transplanted hepatocyte nodules. The statisticalsignificance of the reductions in the viral replication levels aredetermined.

Another example of a mouse model of HCV involves implantation of theHuH7 cell line expressing a luciferase reporter linked to the HCVsubgenome into SCID mice, subcutaneously or directly into the liver (SeeZhu et al., Antimicrobial Agents and Chemother., 2006, 50:3260-3268).The mice are treated with a compound or placebo, and whole-body imagingis used to detect and quantify bioluminescence signal intensity. Micetreated with a compound that is effective against HCV have lessbioluminescence signal intensity relative to mice treated with placeboor a negative control.

4.5.5 HIV

The safety and efficacy of compounds against HIV can be assessed in vivowith established animal models well known in the art. For example, aTrimera mouse model of HIV-1 infection has been developed byreconstituting irradiated normal BALB/c mice with murine SCID bonemarrow and engrafted human peripheral blood mononuclear cells (SeeAyash-Rashkovsky et al., FASEB J., 2005, 19:1149-1151). These mice areinjected intraperitoneally with T- and M-tropic HIV-1 laboratorystrains. After HIV infection, rapid loss of human CD4⁺ T cells, decreasein CD4/CD8 ratio, and increased T cell activation can be observed. Acompound can be administered to these mice and standard assays known inthe art can be used to determine the viral replication capacity inanimals treated or untreated with a compound. Non-limiting examples ofsuch assays include the COBAS AMPLICOR® RT-PCR assay (Roche Diagnostics,Branchberg, N.J.) to determine plasma viral load (HIV-1 RNA copies/ml);active HIV-1 virus replication assay where human lymphocytes recoveredfrom infected Trimera mice were cocultured with target T cells (MT-2cells) and HIV-dependent syncytia formation was examined; and humanlymphocytes recovered from infected Trimera mice were cocultured withcMAGI indicator cells, where HIV-1 LTR driven trans-activation ofβ-galactosidase was measured. Levels of anti-HIV-1 antibodies producedin these mice can also be measured by ELISA. Other established mousemodels described in the art can also be used to test the antiviralactivity of compounds in vivo (See, Mosier et al., Semin. Immunol.,1996, 8:255-262; Mosier et al., Hosp. Pract. (Off Ed)., 1996, 31:41-48,53-55, 59-60; Bonyhadi et al., Mol. Med. Today, 1997, 3:246-253;Jolicoeur et al., Leukemia, 1999, 13:S78-S80; Browning et al., Proc.Natl. Acad. Sci. USA, 1997, 94:14637-14641; and Sawada et al., J. Exp.Med., 1998, 187:1439-1449). A simian immunodeficiency virus (SIV)nonhuman primate model has also been described (See Schito et al., Curr.HIV Res., 2006, 4:379-386).

5. Pharmaceutical Compositions

Any compound described or incorporated by referenced herein mayoptionally be in the form of a composition comprising the compound.

In certain embodiments provided herein, compositions (includingpharmaceutical compositions) comprise a compound and a pharmaceuticallyacceptable carrier, excipient, or diluent.

In other embodiments, provided herein are pharmaceutical compositionscomprising an effective amount of a compound and a pharmaceuticallyacceptable carrier, excipient, or diluent. The pharmaceuticalcompositions are suitable for veterinary and/or human administration.

The pharmaceutical compositions provided herein can be in any form thatallows for the composition to be administered to a subject, said subjectpreferably being an animal, including, but not limited to a human,mammal, or non-human animal, such as a cow, horse, sheep, pig, fowl,cat, dog, mouse, rat, rabbit, guinea pig, etc., and is more preferably amammal, and most preferably a human.

In a specific embodiment and in this context, the term “pharmaceuticallyacceptable carrier, excipient or diluent” means a carrier, excipient ordiluent approved by a regulatory agency of the Federal or a stategovernment or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly inhumans. The term “carrier” refers to a diluent, adjuvant (e.g., Freund'sadjuvant (complete and incomplete)), excipient, or vehicle with whichthe therapeutic is administered. Such pharmaceutical carriers can besterile liquids, such as water and oils, including those of petroleum,animal, vegetable or synthetic origin, such as peanut oil, soybean oil,mineral oil, sesame oil and the like. Water is a preferred carrier whenthe pharmaceutical composition is administered intravenously. Salinesolutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions.Examples of suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin.

Typical compositions and dosage forms comprise one or more excipients.Suitable excipients are well-known to those skilled in the art ofpharmacy, and non limiting examples of suitable excipients includestarch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, ethanoland the like. Whether a particular excipient is suitable forincorporation into a pharmaceutical composition or dosage form dependson a variety of factors well known in the art including, but not limitedto, the way in which the dosage form will be administered to a patientand the specific active ingredients in the dosage form. The compositionor single unit dosage form, if desired, can also contain minor amountsof wetting or emulsifying agents, or pH buffering agents.

Lactose free compositions can comprise excipients that are well known inthe art and are listed, for example, in the U.S. Pharmacopeia (USP) SP(XXI)/NF (XVI). In general, lactose free compositions comprise an activeingredient, a binder/filler, and a lubricant in pharmaceuticallycompatible and pharmaceutically acceptable amounts. Preferred lactosefree dosage forms comprise a compound, microcrystalline cellulose, pregelatinized starch, and magnesium stearate.

Further provided herein are anhydrous pharmaceutical compositions anddosage forms comprising one or more compounds, since water canfacilitate the degradation of some compounds. For example, the additionof water (e.g., 5%) is widely accepted in the pharmaceutical arts as ameans of simulating long term storage in order to determinecharacteristics such as shelf life or the stability of formulations overtime. See, e.g., Jens T. Carstensen, Drug Stability: Principles &Practice, 2d. Ed., Marcel Dekker, NY, N.Y., 1995, pp. 379 80. In effect,water and heat accelerate the decomposition of some compounds. Thus, theeffect of water on a formulation can be of great significance sincemoisture and/or humidity are commonly encountered during manufacture,handling, packaging, storage, shipment, and use of formulations.

Anhydrous compositions and dosage forms provided herein can be preparedusing anhydrous or low moisture containing ingredients and low moistureor low humidity conditions. Compositions and dosage forms that compriselactose and at least one compound that comprises a primary or secondaryamine are preferably anhydrous if substantial contact with moistureand/or humidity during manufacturing, packaging, and/or storage isexpected.

An anhydrous composition should be prepared and stored such that itsanhydrous nature is maintained. Accordingly, anhydrous compositions arepreferably packaged using materials known to prevent exposure to watersuch that they can be included in suitable formulary kits. Examples ofsuitable packaging include, but are not limited to, hermetically sealedfoils, plastics, unit dose containers (e.g., vials), blister packs, andstrip packs.

Further provided herein are compositions and dosage forms that compriseone or more agents that reduce the rate by which a compound willdecompose. Such agents, which are referred to herein as “stabilizers,”include, but are not limited to, antioxidants such as ascorbic acid, pHbuffers, or salt buffers.

The compositions and single unit dosage forms can take the form ofsolutions, suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations and the like. Oral formulation caninclude standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Such compositions and dosage forms willcontain a prophylactically or therapeutically effective amount of acompound preferably in purified form, together with a suitable amount ofcarrier so as to provide the form for proper administration to thepatient. The formulation should suit the mode of administration. In apreferred embodiment, the compositions or single unit dosage forms aresterile and in suitable form for administration to a subject, preferablyan animal subject, more preferably a mammalian subject, and mostpreferably a human subject.

Compositions provided herein are formulated to be compatible with theintended route of administration. Examples of routes of administrationinclude, but are not limited to, parenteral, e.g., intravenous,intradermal, subcutaneous, oral (e.g., inhalation), intranasal,transdermal (topical), transmucosal, intra-synovial, ophthalmic, andrectal administration. In a specific embodiment, the composition isformulated in accordance with routine procedures as a compositionadapted for intravenous, subcutaneous, intramuscular, oral, intranasal,ophthalmic, or topical administration to human beings. In a preferredembodiment, a composition is formulated in accordance with routineprocedures for subcutaneous administration to human beings. Typically,compositions for intravenous administration are solutions in sterileisotonic aqueous buffer. Where necessary, the composition may alsoinclude a solubilizing agent and a local anesthetic such as lignocaineto ease pain at the site of the injection. Examples of dosage formsinclude, but are not limited to: tablets; caplets; capsules, such assoft elastic gelatin capsules; cachets; troches; lozenges; dispersions;suppositories; ointments; cataplasms (poultices); pastes; powders;dressings; creams; plasters; solutions; patches; aerosols (e.g., nasalsprays or inhalers); gels; liquid dosage forms suitable for oral ormucosal administration to a patient, including suspensions (e.g.,aqueous or non aqueous liquid suspensions, oil in water emulsions, or awater in oil liquid emulsions), solutions, and elixirs; liquid dosageforms suitable for parenteral administration to a patient; and sterilesolids (e.g., crystalline or amorphous solids) that can be reconstitutedto provide liquid dosage forms suitable for parenteral administration toa patient.

The composition, shape, and type of dosage forms of the invention willtypically vary depending on their use.

Generally, the ingredients of compositions provided herein are suppliedeither separately or mixed together in unit dosage form, for example, asa dry lyophilized powder or water free concentrate in a hermeticallysealed container such as an ampoule or sachette indicating the quantityof active agent. Where the composition is to be administered byinfusion, it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

Pharmaceutical compositions provided herein that are suitable for oraladministration can be presented as discrete dosage forms, such as, butare not limited to, tablets (e.g., chewable tablets), caplets, capsules,and liquids (e.g., flavored syrups). Such dosage forms containpredetermined amounts of active ingredients, and may be prepared bymethods of pharmacy well known to those skilled in the art. Seegenerally, Remington's Pharmaceutical Sciences, 18th ed., MackPublishing, Easton Pa. (1990).

Typical oral dosage forms provided herein are prepared by combining acompound in an intimate admixture with at least one excipient accordingto conventional pharmaceutical compounding techniques. Excipients cantake a wide variety of forms depending on the form of preparationdesired for administration. For example, excipients suitable for use inoral liquid or aerosol dosage forms include, but are not limited to,water, glycols, oils, alcohols, flavoring agents, preservatives, andcoloring agents. Examples of excipients suitable for use in solid oraldosage forms (e.g., powders, tablets, capsules, and caplets) include,but are not limited to, starches, sugars, micro crystalline cellulose,diluents, granulating agents, lubricants, binders, and disintegratingagents.

Because of their ease of administration, tablets and capsules representthe most advantageous oral dosage unit forms, in which case solidexcipients are employed. If desired, tablets can be coated by standardaqueous or nonaqueous techniques. Such dosage forms can be prepared byany of the methods of pharmacy. In general, pharmaceutical compositionsand dosage forms are prepared by uniformly and intimately admixing theactive ingredients with liquid carriers, finely divided solid carriers,or both, and then shaping the product into the desired presentation ifnecessary.

For example, a tablet can be prepared by compression or molding.Compressed tablets can be prepared by compressing in a suitable machinethe active ingredients in a free flowing form such as powder orgranules, optionally mixed with an excipient. Molded tablets can be madeby molding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

Examples of excipients that can be used in oral dosage forms providedherein include, but are not limited to, binders, fillers, disintegrants,and lubricants. Binders suitable for use in pharmaceutical compositionsand dosage forms include, but are not limited to, corn starch, potatostarch, or other starches, gelatin, natural and synthetic gums such asacacia, sodium alginate, alginic acid, other alginates, powderedtragacanth, guar gum, cellulose and its derivatives (e.g., ethylcellulose, cellulose acetate, carboxymethyl cellulose calcium, sodiumcarboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pregelatinized starch, hydroxypropyl methyl cellulose, (e.g., Nos. 2208,2906, 2910), microcrystalline cellulose, and mixtures thereof.

Examples of fillers suitable for use in the pharmaceutical compositionsand dosage forms provided herein include, but are not limited to, talc,calcium carbonate (e.g., granules or powder), microcrystallinecellulose, powdered cellulose, dextrates, kaolin, mannitol, silicicacid, sorbitol, starch, pre gelatinized starch, and mixtures thereof.The binder or filler in pharmaceutical compositions provided herein istypically present in from about 50 to about 99 weight percent of thepharmaceutical composition or dosage form.

Suitable forms of microcrystalline cellulose include, but are notlimited to, the materials sold as AVICEL PH 101, AVICEL PH 103 AVICEL RC581, AVICEL PH 105 (available from FMC Corporation, American ViscoseDivision, Avicel Sales, Marcus Hook, Pa.), and mixtures thereof. Aspecific binder is a mixture of microcrystalline cellulose and sodiumcarboxymethyl cellulose sold as AVICEL RC 581. Suitable anhydrous or lowmoisture excipients or additives include AVICEL PH 103™ and Starch 1500LM.

Disintegrants are used in the compositions provided herein to providetablets that disintegrate when exposed to an aqueous environment.Tablets that contain too much disintegrant may disintegrate in storage,while those that contain too little may not disintegrate at a desiredrate or under the desired conditions. Thus, a sufficient amount ofdisintegrant that is neither too much nor too little to detrimentallyalter the release of the active ingredients should be used to form solidoral dosage forms provided herein. The amount of disintegrant usedvaries based upon the type of formulation, and is readily discernible tothose of ordinary skill in the art. Typical pharmaceutical compositionscomprise from about 0.5 to about 15 weight percent of disintegrant,specifically from about 1 to about 5 weight percent of disintegrant.

Disintegrants that can be used in pharmaceutical compositions and dosageforms provided herein include, but are not limited to, agar, alginicacid, calcium carbonate, microcrystalline cellulose, croscarmellosesodium, crospovidone, polacrilin potassium, sodium starch glycolate,potato or tapioca starch, pre gelatinized starch, other starches, clays,other algins, other celluloses, gums, and mixtures thereof.

Lubricants that can be used in pharmaceutical compositions and dosageforms provided herein include, but are not limited to, calcium stearate,magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol,mannitol, polyethylene glycol, other glycols, stearic acid, sodiumlauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanut oil,cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, andsoybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, andmixtures thereof. Additional lubricants include, for example, a syloidsilica gel (AEROSIL 200, manufactured by W.R. Grace Co. of Baltimore,Md.), a coagulated aerosol of synthetic silica (marketed by Degussa Co.of Plano, Tex.), CAB O SIL (a pyrogenic silicon dioxide product sold byCabot Co. of Boston, Mass.), and mixtures thereof. If used at all,lubricants are typically used in an amount of less than about 1 weightpercent of the pharmaceutical compositions or dosage forms into whichthey are incorporated.

A compound can be administered by controlled release means or bydelivery devices that are well known to those of ordinary skill in theart. Examples include, but are not limited to, those described in U.S.Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; and U.S. Pat. Nos.4,008,719, 5,674,533, 5,059,595, 5,591,767, 5,120,548, 5,073,543,5,639,476, 5,354,556, and 5,733,566, each of which is incorporatedherein by reference. Such dosage forms can be used to provide slow orcontrolled release of one or more active ingredients using, for example,hydropropylmethyl cellulose, other polymer matrices, gels, permeablemembranes, osmotic systems, multilayer coatings, microparticles,liposomes, microspheres, or a combination thereof to provide the desiredrelease profile in varying proportions. Suitable controlled releaseformulations known to those of ordinary skill in the art, includingthose described herein, can be readily selected for use with the activeingredients of the invention. The invention thus encompasses single unitdosage forms suitable for oral administration such as, but not limitedto, tablets, capsules, gelcaps, and caplets that are adapted forcontrolled release.

All controlled release pharmaceutical products have a common goal ofimproving drug therapy over that achieved by their non controlledcounterparts. Ideally, the use of an optimally designed controlledrelease preparation in medical treatment is characterized by a minimumof drug substance being employed to cure or control the condition in aminimum amount of time. Advantages of controlled release formulationsinclude extended activity of the drug, reduced dosage frequency, andincreased patient compliance. In addition, controlled releaseformulations can be used to affect the time of onset of action or othercharacteristics, such as blood levels of the drug, and can thus affectthe occurrence of side (e.g., adverse) effects.

Most controlled release formulations are designed to initially releasean amount of drug (active ingredient) that promptly produces the desiredtherapeutic effect, and gradually and continually release of otheramounts of drug to maintain this level of therapeutic or prophylacticeffect over an extended period of time. In order to maintain thisconstant level of drug in the body, the drug must be released from thedosage form at a rate that will replace the amount of drug beingmetabolized and excreted from the body. Controlled release of an activeingredient can be stimulated by various conditions including, but notlimited to, pH, temperature, enzymes, water, or other physiologicalconditions or agents.

Parenteral dosage forms can be administered to patients by variousroutes including, but not limited to, subcutaneous, intravenous(including bolus injection), intramuscular, and intraarterial. Becausetheir administration typically bypasses patients' natural defensesagainst contaminants, parenteral dosage forms are preferably sterile orcapable of being sterilized prior to administration to a patient.Examples of parenteral dosage forms include, but are not limited to,solutions ready for injection, dry products ready to be dissolved orsuspended in a pharmaceutically acceptable vehicle for injection,suspensions ready for injection, and emulsions.

Suitable vehicles that can be used to provide parenteral dosage formsprovided herein are well known to those skilled in the art. Examplesinclude, but are not limited to: Water for Injection USP; aqueousvehicles such as, but not limited to, Sodium Chloride Injection,Ringer's Injection, Dextrose Injection, Dextrose and Sodium ChlorideInjection, and Lactated Ringer's Injection; water miscible vehicles suchas, but not limited to, ethyl alcohol, polyethylene glycol, andpolypropylene glycol; and non aqueous vehicles such as, but not limitedto, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate,isopropyl myristate, and benzyl benzoate.

Agents that increase the solubility of one or more of the compoundsprovided herein can also be incorporated into the parenteral dosageforms provided herein.

Transdermal, topical, and mucosal dosage forms provided herein include,but are not limited to, ophthalmic solutions, sprays, aerosols, creams,lotions, ointments, gels, solutions, emulsions, suspensions, or otherforms known to one of skill in the art. See, e.g., Remington'sPharmaceutical Sciences, 16th and 18th eds., Mack Publishing, Easton Pa.(1980 & 1990); and Introduction to Pharmaceutical Dosage Forms, 4th ed.,Lea & Febiger, Philadelphia (1985). Dosage forms suitable for treatingmucosal tissues within the oral cavity can be formulated as mouthwashesor as oral gels. Further, transdermal dosage forms include “reservoirtype” or “matrix type” patches, which can be applied to the skin andworn for a specific period of time to permit the penetration of adesired amount of active ingredients.

Suitable excipients (e.g., carriers and diluents) and other materialsthat can be used to provide transdermal, topical, and mucosal dosageforms provided herein are well known to those skilled in thepharmaceutical arts, and depend on the particular tissue to which agiven pharmaceutical composition or dosage form will be applied. Withthat fact in mind, typical excipients include, but are not limited to,water, acetone, ethanol, ethylene glycol, propylene glycol, butane 1,3diol, isopropyl myristate, isopropyl palmitate, mineral oil, andmixtures thereof to form lotions, tinctures, creams, emulsions, gels orointments, which are non toxic and pharmaceutically acceptable.Moisturizers or humectants can also be added to pharmaceuticalcompositions and dosage forms if desired. Examples of such additionalingredients are well known in the art. See, e.g., Remington'sPharmaceutical Sciences, 16th and 18th eds., Mack Publishing, Easton Pa.(1980 & 1990).

Depending on the specific tissue to be treated, additional componentsmay be used prior to, in conjunction with, or subsequent to treatmentwith a compound. For example, penetration enhancers can be used toassist in delivering the active ingredients to the tissue. Suitablepenetration enhancers include, but are not limited to: acetone; variousalcohols such as ethanol, oleyl, and tetrahydrofuryl; alkyl sulfoxidessuch as dimethyl sulfoxide; dimethyl acetamide; dimethyl formamide;polyethylene glycol; pyrrolidones such as polyvinylpyrrolidone; Kollidongrades (Povidone, Polyvidone); urea; and various water soluble orinsoluble sugar esters such as Tween 80 (polysorbate 80) and Span 60(sorbitan monostearate).

The pH of a pharmaceutical composition or dosage form, or of the tissueto which the pharmaceutical composition or dosage form is applied, mayalso be adjusted to improve delivery of one or more compounds.Similarly, the polarity of a solvent carrier, its ionic strength, ortonicity can be adjusted to improve delivery. Agents such as stearatescan also be added to pharmaceutical compositions or dosage forms toadvantageously alter the hydrophilicity or lipophilicity of one or morecompounds so as to improve delivery. In this regard, stearates can serveas a lipid vehicle for the formulation, as an emulsifying agent orsurfactant, and as a delivery enhancing or penetration enhancing agent.Different salts, hydrates or solvates of the compounds can be used tofurther adjust the properties of the resulting composition.

In certain specific embodiments, the compositions are in oral,injectable, or transdermal dosage forms. In one specific embodiment, thecompositions are in oral dosage forms. In another specific embodiment,the compositions are in the form of injectable dosage forms. In anotherspecific embodiment, the compositions are in the form of transdermaldosage forms.

6. Prophylactic and Therapeutic Methods

The present invention provides methods of preventing, treating and/ormanaging a viral infection, said methods comprising administering to asubject in need thereof one or more compounds. In a specific embodiment,the invention provides a method of preventing, treating and/or managinga viral infection, said method comprising administering to a subject inneed thereof a dose of a prophylactically or therapeutically effectiveamount of one or more compounds or a composition comprising a compound.A compound or a composition comprising a compound may be used as anyline of therapy (e.g., a first, second, third, fourth or fifth linetherapy) for a viral infection.

In another embodiment, the invention relates to a method for reversingor redirecting metabolic flux altered by viral infection in a humansubject by administering to a human subject in need thereof, aneffective amount of one or more compounds or a composition comprisingone or more compounds. For example, viral infection can be treated usingcombinations of the enzyme inhibition compounds that produce beneficialresults, e.g., synergistic effect; reduction of side effects; a highertherapeutic index. In one such embodiment, a citrate lyase inhibitor canbe used in combination with an Acetyl-CoA Carboxylase (ACC).

In specific embodiments, a compound is the only active ingredientadministered to prevent, treat, manage or ameliorate said viralinfection. In a certain embodiment, a composition comprising a compoundis the only active ingredient.

The choice of compounds to be used depends on a number of factors,including but not limited to the type of viral infection, health and ageof the patient, and toxicity or side effects. For example, treatmentsthat inhibit enzymes required for core ATP production, such as protonATPase are not preferred unless given in a regimen that compensates forthe toxicity; e.g., using a localized delivery system that limitssystemic distribution of the drug.

The present invention encompasses methods for preventing, treating,and/or managing a viral infection for which no antiviral therapy isavailable. The present invention also encompasses methods forpreventing, treating, and/or managing a viral infection as analternative to other conventional therapies.

The present invention also provides methods of preventing, treatingand/or managing a viral infection, said methods comprising administeringto a subject in need thereof one or more of the compounds and one ormore other therapies (e.g., prophylactic or therapeutic agents). In aspecific embodiment, the other therapies are currently being used, havebeen used or are known to be useful in the prevention, treatment and/ormanagement of a viral infection. Non-limiting examples of such therapiesare provided in Section 6, infra. In a specific embodiment, one or morecompounds are administered to a subject in combination with one or moreof the therapies described in Section 6, infra. In another embodiment,one or more compounds are administered to a subject in combination witha supportive therapy, a pain relief therapy, or other therapy that doesnot have antiviral activity.

The combination therapies of the invention can be administeredsequentially or concurrently. In one embodiment, the combinationtherapies of the invention comprise a compound and at least one othertherapy which has the same mechanism of action. In another embodiment,the combination therapies of the invention comprise a compound and atleast one other therapy which has a different mechanism of action thanthe compound.

In a specific embodiment, the combination therapies of the presentinvention improve the prophylactic and/or therapeutic effect of acompound by functioning together with the compound to have an additiveor synergistic effect. In another embodiment, the combination therapiesof the present invention reduce the side effects associated with eachtherapy taken alone.

The prophylactic or therapeutic agents of the combination therapies canbe administered to a subject in the same pharmaceutical composition.Alternatively, the prophylactic or therapeutic agents of the combinationtherapies can be administered concurrently to a subject in separatepharmaceutical compositions. The prophylactic or therapeutic agents maybe administered to a subject by the same or different routes ofadministration.

6.1 Patient Population

According to the invention, compounds, compositions comprising acompound, or a combination therapy is administered to a subjectsuffering from a viral infection. In other embodiments, compounds,compositions comprising a compound, or a combination therapy isadministered to a subject predisposed or susceptible to a viralinfection. In some embodiments, compounds, compositions comprising acompound, or a combination therapy is administered to a subject thatlives in a region where there has been or might be an outbreak with aviral infection. In some embodiments, the viral infection is a latentviral infection. In one embodiment, a compound or a combination therapyis administered to a human infant. In one embodiment, a compound or acombination therapy is administered to a premature human infant. Inother embodiments, the viral infection is an active infection. In yetother embodiments, the viral infection is a chronic viral infection.Non-limiting examples of types of virus infections include infectionscaused by those provided in Section 4.1, supra.

In a specific embodiment, the viral infection is an enveloped virusinfection. In some embodiments, the enveloped virus is a DNA virus. Inother embodiments, the enveloped virus is a RNA virus. In someembodiments, the enveloped virus has a double stranded DNA or RNAgenome. In other embodiments, the enveloped virus has a single-strandedDNA or RNA genome. In a specific embodiment, the virus infects humans.

In certain embodiments, a compound, a composition comprising a compound,or a combination therapy is administered to a mammal which is 0 to 6months old, 6 to 12 months old, 1 to 5 years old, 5 to 10 years old, 10to 15 years old, 15 to 20 years old, 20 to 25 years old, 25 to 30 yearsold, 30 to 35 years old, 35 to 40 years old, 40 to 45 years old, 45 to50 years old, 50 to 55 years old, 55 to 60 years old, 60 to 65 yearsold, 65 to 70 years old, 70 to 75 years old, 75 to 80 years old, 80 to85 years old, 85 to 90 years old, 90 to 95 years old or 95 to 100 yearsold. In certain embodiments, a compound, a composition comprising acompound, or a combination therapy is administered to a human at riskfor a virus infection. In certain embodiments, a compound, a compositioncomprising a compound, or a combination therapy is administered to ahuman with a virus infection. In certain embodiments, the patient is ahuman 0 to 6 months old, 6 to 12 months old, 1 to 5 years old, 5 to 10years old, 5 to 12 years old, 10 to 15 years old, 15 to 20 years old, 13to 19 years old, 20 to 25 years old, 25 to 30 years old, 20 to 65 yearsold, 30 to 35 years old, 35 to 40 years old, 40 to 45 years old, 45 to50 years old, 50 to 55 years old, 55 to 60 years old, 60 to 65 yearsold, 65 to 70 years old, 70 to 75 years old, 75 to 80 years old, 80 to85 years old, 85 to 90 years old, 90 to 95 years old or 95 to 100 yearsold. In some embodiments, a compound, a composition comprising acompound, or a combination therapy is administered to a human infant. Inother embodiments, a compound, or a combination therapy is administeredto a human child. In other embodiments, a compound, a compositioncomprising a compound, or a combination therapy is administered to ahuman adult. In yet other embodiments, a compound, a compositioncomprising a compound, or a combination therapy is administered to anelderly human.

In certain embodiments, a compound, a composition comprising a compound,or a combination therapy is administered to a pet, e.g., a dog or cat.In certain embodiments, a compound, a composition comprising a compound,or a combination therapy is administered to a farm animal or livestock,e.g., pig, cows, horses, chickens, etc. In certain embodiments, acompound, a composition comprising a compound, or a combination therapyis administered to a bird, e.g., ducks or chicken.

In certain embodiments, a compound, a composition comprising a compound,or a combination therapy is administered to a primate, preferably ahuman, or another mammal, such as a pig, cow, horse, sheep, goat, dog,cat and rodent, in an immunocompromised state or immunosuppressed stateor at risk for becoming immunocompromised or immunosuppressed. Incertain embodiments, a compound, a composition comprising a compound, ora combination therapy is administered to a subject receiving orrecovering from immunosuppressive therapy. In certain embodiments, acompound, a composition comprising a compound, or a combination therapyis administered to a subject that has or is at risk of getting cancer,AIDS, another viral infection, or a bacterial infection. In certainembodiments, a subject that is, will or has undergone surgery,chemotherapy and/or radiation therapy. In certain embodiments, acompound, a composition comprising a compound, or a combination therapyis administered to a subject that has cystic fibrosis, pulmonaryfibrosis, or another disease which makes the subject susceptible to aviral infection. In certain embodiments, a compound, a compositioncomprising a compound, or a combination therapy is administered to asubject that has, will have or had a tissue transplant. In someembodiments, a compound, a composition comprising a compound, or acombination therapy is administered to a subject that lives in a nursinghome, a group home (i.e., a home for 10 or more subjects), or a prison.In some embodiments, a compound, a composition comprising a compound, ora combination therapy is administered to a subject that attends school(e.g., elementary school, middle school, junior high school, high schoolor university) or daycare. In some embodiments, a compound, acomposition comprising a compound, or a combination therapy isadministered to a subject that works in the healthcare area, such as adoctor or a nurse, or in a hospital. In certain embodiments, a compound,a composition comprising a compound, or a combination therapy isadministered to a subject that is pregnant or will become pregnant.

In some embodiments, a patient is administered a compound or acomposition comprising a compound, or a combination therapy before anyadverse effects or intolerance to therapies other than compoundsdevelops. In some embodiments, compounds or compositions comprising oneor more compounds, or combination therapies are administered torefractory patients. In a certain embodiment, refractory patient is apatient refractory to a standard antiviral therapy. In certainembodiments, a patient with a viral infection, is refractory to atherapy when the infection has not significantly been eradicated and/orthe symptoms have not been significantly alleviated. The determinationof whether a patient is refractory can be made either in vivo or invitro by any method known in the art for assaying the effectiveness of atreatment of infections, using art-accepted meanings of “refractory” insuch a context. In various embodiments, a patient with a viral infectionis refractory when viral replication has not decreased or has increased.

In some embodiments, compounds or compositions comprising one or morecompounds, or combination therapies are administered to a patient toprevent the onset or reoccurrence of viral infections in a patient atrisk of developing such infections. In some embodiments, compounds orcompositions comprising one or more compounds, or combination therapiesare administered to a patient who are susceptible to adverse reactionsto conventional therapies.

In some embodiments, one or more compounds or compositions comprisingone or more compounds, or combination therapies are administered to apatient who has proven refractory to therapies other than compounds, butare no longer on these therapies. In certain embodiments, the patientsbeing managed or treated in accordance with the methods of thisinvention are patients already being treated with antibiotics,anti-virals, anti-fungals, or other biological therapy/immunotherapy.Among these patients are refractory patients, patients who are too youngfor conventional therapies, and patients with reoccurring viralinfections despite management or treatment with existing therapies.

In some embodiments, the subject being administered one or morecompounds or compositions comprising one or more compounds, orcombination therapies has not received a therapy prior to theadministration of the compounds or compositions or combinationtherapies. In other embodiments, one or more compounds or compositionscomprising one or more compounds, or combination therapies areadministered to a subject who has received a therapy prior toadministration of one or more compounds or compositions comprising oneor more compounds, or combination therapies. In some embodiments, thesubject administered a compound or a composition comprising a compoundwas refractory to a prior therapy or experienced adverse side effects tothe prior therapy or the prior therapy was discontinued due tounacceptable levels of toxicity to the subject.

6.2 Mode of Administration

When administered to a patient, a compound is preferably administered asa component of a composition that optionally comprises apharmaceutically acceptable vehicle. The composition can be administeredorally, or by any other convenient route, for example, by infusion orbolus injection, by absorption through epithelial or mucocutaneouslinings (e.g., oral mucosa, rectal, and intestinal mucosa) and may beadministered together with another biologically active agent.Administration can be systemic or local. Various delivery systems areknown, e.g., encapsulation in liposomes, microparticles, microcapsules,capsules, and can be used to administer the compound andpharmaceutically acceptable salts thereof.

Methods of administration include but are not limited to parenteral,intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous,intranasal, epidural, oral, sublingual, intranasal, intracerebral,intravaginal, transdermal, rectally, by inhalation, or topically,particularly to the ears, nose, eyes, or skin. The mode ofadministration is left to the discretion of the practitioner. In mostinstances, administration will result in the release of a compound intothe bloodstream.

In specific embodiments, it may be desirable to administer a compoundlocally. This may be achieved, for example, and not by way oflimitation, by local infusion, topical application, e.g., in conjunctionwith a wound dressing, by injection, by means of a catheter, by means ofa suppository, or by means of an implant, said implant being of aporous, non-porous, or gelatinous material, including membranes, such assialastic membranes, or fibers. In such instances, administration mayselectively target a local tissue without substantial release of aCompound into the bloodstream.

In certain embodiments, it may be desirable to introduce a compound intothe central nervous system by any suitable route, includingintraventricular, intrathecal and epidural injection. Intraventricularinjection may be facilitated by an intraventricular catheter, forexample, attached to a reservoir, such as an Ommaya reservoir.

Pulmonary administration can also be employed, e.g., by use of aninhaler or nebulizer, and formulation with an aerosolizing agent, or viaperfusion in a fluorocarbon or synthetic pulmonary surfactant. Incertain embodiments, a compound is formulated as a suppository, withtraditional binders and vehicles such as triglycerides.

For viral infections with cutaneous manifestations, the compound can beadministered topically. Similarly, for viral infections with ocularmanifestation, the compounds can be administered ocularly.

In another embodiment, a compound is delivered in a vesicle, inparticular a liposome (See Langer, 1990, Science 249:1527 1533; Treat etal., in Liposomes in the Therapy of Infectious Disease and Bacterialinfection, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353365 (1989); Lopez Berestein, ibid., pp. 317 327; See generally ibid.).

In another embodiment, a compound is delivered in a controlled releasesystem (See, e.g., Goodson, in Medical Applications of ControlledRelease, supra, vol. 2, pp. 115 138 (1984)). Examples ofcontrolled-release systems are discussed in the review by Langer, 1990,Science 249:1527 1533 may be used. In one embodiment, a pump may be used(See Langer, supra; Sefton, 1987, CRC Crit. Ref Biomed. Eng. 14:201;Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J.Med. 321:574). In another embodiment, polymeric materials can be used(See Medical Applications of Controlled Release, Langer and Wise (eds.),CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, NewYork (1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol.Chem. 23:61; See also Levy et al., 1985, Science 228:190; During et al.,1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105).In a specific embodiment, a controlled-release system comprising acompound is placed in close proximity to the tissue infected with avirus to be prevented, treated and/or managed. In accordance with thisembodiment, the close proximity of the controlled-release system to theinfection may result in only a fraction of the dose of the compoundrequired if it is systemically administered.

In certain embodiments, it may be preferable to administer a compoundvia the natural route of infection of the virus against which a compoundhas antiviral activity. For example, it may be desirable to administer acompound of the invention into the lungs by any suitable route to treator prevent an infection of the respiratory tract by viruses (e.g.,influenza virus). Pulmonary administration can also be employed, e.g.,by use of an inhaler or nebulizer, and formulation with an aerosolizingagent for use as a spray.

6.3 Agents for Use in Combination with Compounds

Therapeutic or prophylactic agents that can be used in combination withcompounds for the prevention, treatment and/or management of a viralinfection include, but are not limited to, small molecules, syntheticdrugs, peptides (including cyclic peptides), polypeptides, proteins,nucleic acids (e.g., DNA and RNA nucleotides including, but not limitedto, antisense nucleotide sequences, triple helices, RNAi, and nucleotidesequences encoding biologically active proteins, polypeptides orpeptides), antibodies, synthetic or natural inorganic molecules, mimeticagents, and synthetic or natural organic molecules. Specific examples ofsuch agents include, but are not limited to, immunomodulatory agents(e.g., interferon), anti-inflammatory agents (e.g., adrenocorticoids,corticosteroids (e.g., beclomethasone, budesonide, flunisolide,fluticasone, triamcinolone, methylprednisolone, prednisolone,prednisone, hydrocortisone), glucocorticoids, steriods, andnon-steriodal anti-inflammatory drugs (e.g., aspirin, ibuprofen,diclofenac, and COX-2 inhibitors), pain relievers, leukotreineantagonists (e.g., montelukast, methyl xanthines, zafirlukast, andzileuton), beta2-agonists (e.g., albuterol, biterol, fenoterol,isoetharie, metaproterenol, pirbuterol, salbutamol, terbutalinformoterol, salmeterol, and salbutamol terbutaline), anticholinergicagents (e.g., ipratropium bromide and oxitropium bromide),sulphasalazine, penicillamine, dapsone, antihistamines, anti-malarialagents (e.g., hydroxychloroquine), anti-viral agents (e.g., nucleosideanalogs (e.g., zidovudine, acyclovir, gancyclovir, vidarabine,idoxuridine, trifluridine, and ribavirin), foscarnet, amantadine,rimantadine, saquinavir, indinavir, ritonavir, and AZT) and antibiotics(e.g., dactinomycin (formerly actinomycin), bleomycin, erythomycin,penicillin, mithramycin, and anthramycin (AMC)).

Any therapy which is known to be useful, or which has been used or iscurrently being used for the prevention, management, and/or treatment ofa viral infection or can be used in combination with compounds inaccordance with the invention described herein. See, e.g., Gilman etal., Goodman and Gilman's: The Pharmacological Basis of Therapeutics,10th ed., McGraw-Hill, New York, 2001; The Merck Manual of Diagnosis andTherapy, Berkow, M. D. et al. (eds.), 17th Ed., Merck Sharp & DohmeResearch Laboratories, Rahway, N J, 1999; Cecil Textbook of Medicine,20th Ed., Bennett and Plum (eds.), W.B. Saunders, Philadelphia, 1996,and Physicians' Desk Reference (61^(st) ed. 1007) for informationregarding therapies (e.g., prophylactic or therapeutic agents) whichhave been or are currently being used for preventing, treating and/ormanaging viral infections.

6.3.1 Antiviral Agents

Antiviral agents that can be used in combination with compounds include,but are not limited to, non-nucleoside reverse transcriptase inhibitors,nucleoside reverse transcriptase inhibitors, protease inhibitors, andfusion inhibitors. In one embodiment, the antiviral agent is selectedfrom the group consisting of amantadine, oseltamivir phosphate,rimantadine, and zanamivir. In another embodiment, the antiviral agentis a non-nucleoside reverse transcriptase inhibitor selected from thegroup consisting of delavirdine, efavirenz, and nevirapine. In anotherembodiment, the antiviral agent is a nucleoside reverse transcriptaseinhibitor selected from the group consisting of abacavir, didanosine,emtricitabine, emtricitabine, lamivudine, stavudine, tenofovir DF,zalcitabine, and zidovudine. In another embodiment, the antiviral agentis a protease inhibitor selected from the group consisting ofamprenavir, atazanavir, fosamprenav, indinavir, lopinavir, nelfinavir,ritonavir, and saquinavir. In another embodiment, the antiviral agent isa fusion inhibitor such as enfuvirtide.

Additional, non-limiting examples of antiviral agents for use incombination compounds include the following: rifampicin, nucleosidereverse transcriptase inhibitors (e.g., AZT, ddI, ddC, 3TC, d4T),non-nucleoside reverse transcriptase inhibitors (e.g., delavirdineefavirenz, nevirapine), protease inhibitors (e.g., aprenavir, indinavir,ritonavir, and saquinavir), idoxuridine, cidofovir, acyclovir,ganciclovir, zanamivir, amantadine, and palivizumab. Other examples ofanti-viral agents include but are not limited to acemannan; acyclovir;acyclovir sodium; adefovir; alovudine; alvircept sudotox; amantadinehydrochloride (SYMMETREL™); aranotin; arildone; atevirdine mesylate;avridine; cidofovir; cipamfylline; cytarabine hydrochloride; delavirdinemesylate; desciclovir; didanosine; disoxaril; edoxudine; enviradene;enviroxime; famciclovir; famotine hydrochloride; fiacitabine;fialuridine; fosarilate; foscamet sodium; fosfonet sodium; ganciclovir;ganciclovir sodium; idoxuridine; kethoxal; lamivudine; lobucavir;memotine hydrochloride; methisazone; nevirapine; oseltamivir phosphate(TAMIFLU™); penciclovir; pirodavir; ribavirin; rimantadine hydrochloride(FLUMADINE™); saquinavir mesylate; somantadine hydrochloride;sorivudine; statolon; stavudine; tilorone hydrochloride; trifluridine;valacyclovir hydrochloride; vidarabine; vidarabine phosphate; vidarabinesodium phosphate; viroxime; zalcitabine; zanamivir (RELENZA™);zidovudine; and zinviroxime.

6.3.2 Antibacterial Agents

Antibacterial agents, including antibiotics, that can be used incombination with compounds include, but are not limited to,aminoglycoside antibiotics, glycopeptides, amphenicol antibiotics,ansamycin antibiotics, cephalosporins, cephamycins oxazolidinones,penicillins, quinolones, streptogamins, tetracycline, and analogsthereof. In some embodiments, antibiotics are administered incombination with a compound to prevent and/or treat a bacterialinfection.

In a specific embodiment, compounds are used in combination with otherprotein synthesis inhibitors, including but not limited to,streptomycin, neomycin, erythromycin, carbomycin, and spiramycin.

In one embodiment, the antibacterial agent is selected from the groupconsisting of ampicillin, amoxicillin, ciprofloxacin, gentamycin,kanamycin, neomycin, penicillin G, streptomycin, sulfanilamide, andvancomycin. In another embodiment, the antibacterial agent is selectedfrom the group consisting of azithromycin, cefonicid, cefotetan,cephalothin, cephamycin, chlortetracycline, clarithromycin, clindamycin,cycloserine, dalfopristin, doxycycline, erythromycin, linezolid,mupirocin, oxytetracycline, quinupristin, rifampin, spectinomycin, andtrimethoprim.

Additional, non-limiting examples of antibacterial agents for use incombination with compounds include the following: aminoglycosideantibiotics (e.g., apramycin, arbekacin, bambermycins, butirosin,dibekacin, neomycin, neomycin, undecylenate, netilmicin, paromomycin,ribostamycin, sisomicin, and spectinomycin), amphenicol antibiotics(e.g., azidamfenicol, chloramphenicol, florfenicol, and thiamphenicol),ansamycin antibiotics (e.g., rifamide and rifampin), carbacephems (e.g.,loracarbef), carbapenems (e.g., biapenem and imipenem), cephalosporins(e.g., cefaclor, cefadroxil, cefamandole, cefatrizine, cefazedone,cefozopran, cefpimizole, cefpiramide, and cefpirome), cephamycins (e.g.,cefbuperazone, cefmetazole, and cefminox), folic acid analogs (e.g.,trimethoprim), glycopeptides (e.g., vancomycin), lincosamides (e.g.,clindamycin, and lincomycin), macrolides (e.g., azithromycin,carbomycin, clarithomycin, dirithromycin, erythromycin, and erythromycinacistrate), monobactams (e.g., aztreonam, carumonam, and tigemonam),nitrofurans (e.g., furaltadone, and furazolium chloride), oxacephems(e.g., flomoxef, and moxalactam), oxazolidinones (e.g., linezolid),penicillins (e.g., amdinocillin, amdinocillin pivoxil, amoxicillin,bacampicillin, benzylpenicillinic acid, benzylpenicillin sodium,epicillin, fenbenicillin, floxacillin, penamccillin, penethamatehydriodide, penicillin o benethamine, penicillin 0, penicillin V,penicillin V benzathine, penicillin V hydrabamine, penimepicycline, andphencihicillin potassium), quinolones and analogs thereof (e.g.,cinoxacin, ciprofloxacin, clinafloxacin, flumequine, grepagloxacin,levofloxacin, and moxifloxacin), streptogramins (e.g., quinupristin anddalfopristin), sulfonamides (e.g., acetyl sulfamethoxypyrazine,benzylsulfamide, noprylsulfamide, phthalylsulfacetamide,sulfachrysoidine, and sulfacytine), sulfones (e.g., diathymosulfone,glucosulfone sodium, and solasulfone), and tetracyclines (e.g.,apicycline, chlortetracycline, clomocycline, and demeclocycline).Additional examples include cycloserine, mupirocin, tuberin amphomycin,bacitracin, capreomycin, colistin, enduracidin, enviomycin, and 2,4diaminopyrimidines (e.g., brodimoprim).

6.4 Dosages & Frequency of Administration

The amount of a compound, or the amount of a composition comprising acompound, that will be effective in the prevention, treatment and/ormanagement of a viral infection can be determined by standard clinicaltechniques. In vitro or in vivo assays may optionally be employed tohelp identify optimal dosage ranges. The precise dose to be employedwill also depend, e.g., on the route of administration, the type ofinvention, and the seriousness of the infection, and should be decidedaccording to the judgment of the practitioner and each patient's orsubject's circumstances.

In some embodiments, the dosage of a compound is determined byextrapolating from the no observed adverse effective level (NOAEL), asdetermined in animal studies. This extrapolated dosage is useful indetermining the maximum recommended starting dose for human clinicaltrials. For instance, the NOAELs can be extrapolated to determine humanequivalent dosages (HED). Typically, HED is extrapolated from anon-human animal dosage based on the doses that are normalized to bodysurface area (i.e., mg/m²). In specific embodiments, the NOAELs aredetermined in mice, hamsters, rats, ferrets, guinea pigs, rabbits, dogs,primates, primates (monkeys, marmosets, squirrel monkeys, baboons),micropigs or minipigs. For a discussion on the use of NOAELs and theirextrapolation to determine human equivalent doses, See Guidance forIndustry Estimating the Maximum Safe Starting Dose in Initial ClinicalTrials for Therapeutics in Adult Healthy Volunteers, U.S. Department ofHealth and Human Services Food and Drug Administration Center for DrugEvaluation and Research (CDER), Pharmacology and Toxicology, July 2005.In one embodiment, a compound or composition thereof is administered ata dose that is lower than the human equivalent dosage (HED) of the NOAELover a period of 1 week, 2 weeks, 3 weeks, 1 month, 2 months, threemonths, four months, six months, nine months, 1 year, 2 years, 3 years,4 years or more.

In certain embodiments, a dosage regime for a human subject can beextrapolated from animal model studies using the dose at which 10% ofthe animals die (LD10). In general the starting dose of a Phase Iclinical trial is based on preclinical testing. A standard measure oftoxicity of a drug in preclinical testing is the percentage of animalsthat die because of treatment. It is well within the skill of the art tocorrelate the LD10 in an animal study with the maximal-tolerated dose(MTD) in humans, adjusted for body surface area, as a basis toextrapolate a starting human dose. In some embodiments, theinterrelationship of dosages for one animal model can be converted foruse in another animal, including humans, using conversion factors (basedon milligrams per meter squared of body surface) as described, e.g., inFreireich et al., Cancer Chemother. Rep., 1966, 50:219-244. Body surfacearea may be approximately determined from height and weight of thepatient. See, e.g., Scientific Tables, Geigy Pharmaceuticals, Ardley, N.Y., 1970, 537. In certain embodiments, the adjustment for body surfacearea includes host factors such as, for example, surface area, weight,metabolism, tissue distribution, absorption rate, and excretion rate. Inaddition, the route of administration, excipient usage, and the specificdisease or virus to target are also factors to consider. In oneembodiment, the standard conservative starting dose is about 1/10 themurine LD10, although it may be even lower if other species (i.e., dogs)were more sensitive to the compound. In other embodiments, the standardconservative starting dose is about 1/100, 1/95, 1/90, 1/85, 1/80, 1/75,1/70, 1/65, 1/60, 1/55, 1/50, 1/45, 1/40, 1/35, 1/30, 1/25, 1/20, 1/15,2/10, 3/10, 4/10, or 5/10 of the murine LD10. In other embodiments, anstarting dose amount of a compound in a human is lower than the doseextrapolated from animal model studies. In another embodiment, anstarting dose amount of a compound in a human is higher than the doseextrapolated from animal model studies. It is well within the skill ofthe art to start doses of the active composition at relatively lowlevels, and increase or decrease the dosage as necessary to achieve thedesired effect with minimal toxicity.

Exemplary doses of compounds or compositions include milligram ormicrogram amounts per kilogram of subject or sample weight (e.g., about1 microgram per kilogram to about 500 milligrams per kilogram, about 5micrograms per kilogram to about 100 milligrams per kilogram, or about 1microgram per kilogram to about 50 micrograms per kilogram). In specificembodiments, a daily dose is at least 50 mg, 75 mg, 100 mg, 150 mg, 250mg, 500 mg, 750 mg, or at least 1 g.

In one embodiment, the dosage is a concentration of 0.01 to 5000 mM, 1to 300 mM, 10 to 100 mM and 10 mM to 1 M. In another embodiment, thedosage is a concentration of at least 5 μM, at least 10 μM, at least 50μM, at least 100 μM, at least 500 μM, at least 1 mM, at least 5 mM, atleast 10 mM, at least 50 mM, at least 100 mM, or at least 500 mM.

In one embodiment, the dosage is a concentration of 0.01 to 5000 mM, 1to 300 mM, 10 to 100 mM and 10 mM to 1 M. In another embodiment, thedosage is a concentration of at least 5 μM, at least 10 μM, at least 50μM, at least 100 μM, at least 500 μM, at least 1 mM, at least 5 mM, atleast 10 mM, at least 50 mM, at least 100 mM, or at least 500 mM. In aspecific embodiment, the dosage is 0.25 μg/kg or more, preferably 0.5μg/kg or more, 1 μg/kg or more, 2 μg/kg or more, 3 μg/kg or more, 4μg/kg or more, 5 μg/kg or more, 6 μg/kg or more, 7 μg/kg or more, 8μg/kg or more, 9 μg/kg or more, or 10 μg/kg or more, 25 μg/kg or more,preferably 50 μg/kg or more, 100 μg/kg or more, 250 μg/kg or more, 500μg/kg or more, 1 mg/kg or more, 5 mg/kg or more, 6 mg/kg or more, 7mg/kg or more, 8 mg/kg or more, 9 mg/kg or more, or 10 mg/kg or more ofa patient's body weight.

In another embodiment, the dosage is a unit dose of 5 mg, preferably 10mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 500mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg or more. In anotherembodiment, the dosage is a unit dose that ranges from about 5 mg toabout 100 mg, about 100 mg to about 200 μg, about 150 mg to about 300mg, about 150 mg to about 400 mg, 250 μg to about 500 mg, about 500 mgto about 800 mg, about 500 mg to about 1000 mg, or about 5 mg to about1000 mg.

In certain embodiments, suitable dosage ranges for oral administrationare about 0.001 milligram to about 500 milligrams of a compound, perkilogram body weight per day. In specific embodiments of the invention,the oral dose is about 0.01 milligram to about 100 milligrams perkilogram body weight per day, about 0.1 milligram to about 75 milligramsper kilogram body weight per day or about 0.5 milligram to 5 milligramsper kilogram body weight per day. The dosage amounts described hereinrefer to total amounts administered; that is, if more than one compoundis administered, then, in some embodiments, the dosages correspond tothe total amount administered. In a specific embodiment, oralcompositions contain about 10% to about 95% a compound of the inventionby weight.

Suitable dosage ranges for intravenous (i.v.) administration are about0.01 milligram to about 100 milligrams per kilogram body weight per day,about 0.1 milligram to about 35 milligrams per kilogram body weight perday, and about 1 milligram to about 10 milligrams per kilogram bodyweight per day. In some embodiments, suitable dosage ranges forintranasal administration are about 0.01 pg/kg body weight per day toabout 1 mg/kg body weight per day. Suppositories generally contain about0.01 milligram to about 50 milligrams of a compound of the invention perkilogram body weight per day and comprise active ingredient in the rangeof about 0.5% to about 10% by weight.

Recommended dosages for intradermal, intramuscular, intraperitoneal,subcutaneous, epidural, sublingual, intracerebral, intravaginal,transdermal administration or administration by inhalation are in therange of about 0.001 milligram to about 500 milligrams per kilogram ofbody weight per day. Suitable doses for topical administration includedoses that are in the range of about 0.001 milligram to about 50milligrams, depending on the area of administration. Effective doses maybe extrapolated from dose-response curves derived from in vitro oranimal model test systems. Such animal models and systems are well knownin the art.

In another embodiment, a subject is administered one or more doses of aprophylactically or therapeutically effective amount of a compound or acomposition, wherein the prophylactically or therapeutically effectiveamount is not the same for each dose. In another embodiment, a subjectis administered one or more doses of a prophylactically ortherapeutically effective amount of a compound or a composition, whereinthe dose of a prophylactically or therapeutically effective amountadministered to said subject is increased by, e.g., 0.01 μg/kg, 0.02μg/kg, 0.04 μg/kg, 0.05 μg/kg, 0.06 μg/kg, 0.08 μg/kg, 0.1 μg/kg, 0.2μg/kg, 0.25 μg/kg, 0.5 μg/kg, 0.75 μg/kg, 1 μg/kg, 1.5 μg/kg, 2 μg/kg, 4μg/kg, 5 μg/kg, 10 μg/kg, 15 μg/kg, 20 μg/kg, 25 μg/kg, 30 μg/kg, 35μg/kg, 40 μg/kg, 45 μg/kg, or 50 μg/kg, as treatment progresses. Inanother embodiment, a subject is administered one or more doses of aprophylactically or therapeutically effective amount of a compound orcomposition, wherein the dose is decreased by, e.g., 0.01 μg/kg, 0.02μg/kg, 0.04 μg/kg, 0.05 μg/kg, 0.06 μg/kg, 0.08 μg/kg, 0.1 μg/kg, 0.2μg/kg, 0.25 μg/kg, 0.5 μg/kg, 0.75 μg/kg, 1 μg/kg, 1.5 μg/kg, 2 μg/kg, 4μg/kg, 5 μg/kg, 10 μg/kg, 15 μg/kg, 20 μg/kg, 25 μg/kg, 30 μg/kg, 35μg/kg, 40 μg/kg, 45 μg/kg, or 50 μg/kg, as treatment progresses.

In certain embodiments, a subject is administered a compound or acomposition in an amount effective to inhibit or reduce viral genomereplication by at least 20% to 25%, preferably at least 25% to 30%, atleast 30% to 35%, at least 35% to 40%, at least 40% to 45%, at least 45%to 50%, at least 50% to 55%, at least 55% to 60%, at least 60% to 65%,at least 65% to 70%, at least 70% to 75%, at least 75% to 80%, or up toat least 85% relative to a negative control as determined using an assaydescribed herein or others known to one of skill in the art. In otherembodiments, a subject is administered a compound or a composition in anamount effective to inhibit or reduce viral genome replication by atleast 20% to 25%, preferably at least 25% to 30%, at least 30% to 35%,at least 35% to 40%, at least 40% to 45%, at least 45% to 50%, at least50% to 55%, at least 55% to 60%, at least 60% to 65%, at least 65% to70%, at least 70% to 75%, at least 75% to 80%, or up to at least 85%relative to a negative control as determined using an assay describedherein or others known to one of skill in the art. In certainembodiments, a subject is administered a compound or a composition in anamount effective to inhibit or reduce viral genome replication by atleast 1.5 fold, 2 fold, 2.5 fold, 3 fold, 4 fold, 5 fold, 8 fold, 10fold, 15 fold, 20 fold, or 2 to 5 fold, 2 to 10 fold, 5 to 10 fold, or 5to 20 fold relative to a negative control as determined using an assaydescribed herein or other known to one of skill in the art.

In certain embodiments, a subject is administered a compound or acomposition in an amount effective to inhibit or reduce viral proteinsynthesis by at least 20% to 25%, preferably at least 25% to 30%, atleast 30% to 35%, at least 35% to 40%, at least 40% to 45%, at least 45%to 50%, at least 50% to 55%, at least 55% to 60%, at least 60% to 65%,at least 65% to 70%, at least 70% to 75%, at least 75% to 80%, or up toat least 85% relative to a negative control as determined using an assaydescribed herein or others known to one of skill in the art. In otherembodiments, a subject is administered a compound or a composition in anamount effective to inhibit or reduce viral protein synthesis by atleast 20% to 25%, preferably at least 25% to 30%, at least 30% to 35%,at least 35% to 40%, at least 40% to 45%, at least 45% to 50%, at least50% to 55%, at least 55% to 60%, at least 60% to 65%, at least 65% to70%, at least 70% to 75%, at least 75% to 80%, or up to at least 85%relative to a negative control as determined using an assay describedherein or others known to one of skill in the art. In certainembodiments, a subject is administered a compound or a composition in anamount effective to inhibit or reduce viral protein synthesis by atleast 1.5 fold, 2 fold, 2.5 fold, 3 fold, 4 fold, 5 fold, 8 fold, 10fold, 15 fold, 20 fold, or 2 to 5 fold, 2 to 10 fold, 5 to 10 fold, or 5to 20 fold relative to a negative control as determined using an assaydescribed herein or others known to one of skill in the art.

In certain embodiments, a subject is administered a compound or acomposition in an amount effective to inhibit or reduce viral infectionby at least 20% to 25%, preferably at least 25% to 30%, at least 30% to35%, at least 35% to 40%, at least 40% to 45%, at least 45% to 50%, atleast 50% to 55%, at least 55% to 60%, at least 60% to 65%, at least 65%to 70%, at least 70% to 75%, at least 75% to 80%, or up to at least 85%relative to a negative control as determined using an assay describedherein or others known to one of skill in the art. In some embodiments,a subject is administered a compound or a composition in an amounteffective to inhibit or reduce viral infection by at least 1.5 fold, 2fold, 2.5 fold, 3 fold, 4 fold, 5 fold, 8 fold, 10 fold, 15 fold, 20fold, or 2 to 5 fold, 2 to 10 fold, 5 to 10 fold, or 5 to 20 foldrelative to a negative control as determined using an assay describedherein or others known to one of skill in the art.

In certain embodiments, a subject is administered a compound or acomposition in an amount effective to inhibit or reduce viralreplication by at least 20% to 25%, preferably at least 25% to 30%, atleast 30% to 35%, at least 35% to 40%, at least 40% to 45%, at least 45%to 50%, at least 50% to 55%, at least 55% to 60%, at least 60% to 65%,at least 65% to 70%, at least 70% to 75%, at least 75% to 80%, or up toat least 85% relative to a negative control as determined using an assaydescribed herein or others known to one of skill in the art. In someembodiments, a subject is administered a compound or a composition in anamount effective to inhibit or reduce viral replication by at least 1.5fold, 2 fold, 2.5 fold, 3 fold, 4 fold, 5 fold, 8 fold, 10 fold, 15fold, 20 fold, or 2 to 5 fold, 2 to 10 fold, 5 to 10 fold, or 5 to 20fold relative to a negative control as determined using an assaydescribed herein or others known to one of skill in the art. In otherembodiments, a subject is administered a compound or a composition in anamount effective to inhibit or reduce viral replication by 1 log, 1.5logs, 2 logs, 2.5 logs, 3 logs, 3.5 logs, 4 logs, 5 logs or morerelative to a negative control as determined using an assay describedherein or others known to one of skill in the art.

In certain embodiments, a subject is administered a compound or acomposition in an amount effective to inhibit or reduce the ability ofthe virus to spread to other individuals by at least 20% to 25%,preferably at least 25% to 30%, at least 30% to 35%, at least 35% to40%, at least 40% to 45%, at least 45% to 50%, at least 50% to 55%, atleast 55% to 60%, at least 60% to 65%, at least 65% to 70%, at least 70%to 75%, at least 75% to 80%, or up to at least 85% relative to anegative control as determined using an assay described herein or othersknown to one of skill in the art. In other embodiments, a subject isadministered a compound or a composition in an amount effective toinhibit or reduce the ability of the virus to spread to other cells,tissues or organs in the subject by at least 20% to 25%, preferably atleast 25% to 30%, at least 30% to 35%, at least 35% to 40%, at least 40%to 45%, at least 45% to 50%, at least 50% to 55%, at least 55% to 60%,at least 60% to 65%, at least 65% to 70%, at least 70% to 75%, at least75% to 80%, or up to at least 85% relative to a negative control asdetermined using an assay described herein or others known to one ofskill in the art.

In certain embodiments, a subject is administered a compound or acomposition in an amount effective to inhibit or reduce viral inducedlipid synthesis by at least 20% to 25%, preferably at least 25% to 30%,at least 30% to 35%, at least 35% to 40%, at least 40% to 45%, at least45% to 50%, at least 50% to 55%, at least 55% to 60%, at least 60% to65%, at least 65% to 70%, at least 70% to 75%, at least 75% to 80%, orup to at least 85% relative to a negative control as determined using anassay described herein or others known to one of skill in the art. Inother embodiments, a subject is administered a compound or a compositionin an amount effective to inhibit or reduce viral induced lipidsynthesis by at least 20% to 25%, preferably at least 25% to 30%, atleast 30% to 35%, at least 35% to 40%, at least 40% to 45%, at least 45%to 50%, at least 50% to 55%, at least 55% to 60%, at least 60% to 65%,at least 65% to 70%, at least 70% to 75%, at least 75% to 80%, or up toat least 85% relative to a negative control as determined using an assaydescribed herein or others known to one of skill in the art. In certainembodiments, a subject is administered a compound or a composition in anamount effective to inhibit or reduce viral induced lipid synthesis byat least 1.5 fold, 2 fold, 2.5 fold, 3 fold, 4 fold, 5 fold, 8 fold, 10fold, 15 fold, 20 fold, or 2 to 5 fold, 2 to 10 fold, 5 to 10 fold, or 5to 20 fold relative to a negative control as determined using an assaydescribed herein or others known to one of skill in the art.

In certain embodiments, a dose of a compound or a composition isadministered to a subject every day, every other day, every couple ofdays, every third day, once a week, twice a week, three times a week, oronce every two weeks. In other embodiments, two, three or four doses ofa compound or a composition is administered to a subject every day,every couple of days, every third day, once a week or once every twoweeks. In some embodiments, a dose(s) of a compound or a composition isadministered for 2 days, 3 days, 5 days, 7 days, 14 days, or 21 days. Incertain embodiments, a dose of a compound or a composition isadministered for 1 month, 1.5 months, 2 months, 2.5 months, 3 months, 4months, 5 months, 6 months or more.

The dosages of prophylactic or therapeutic agents which have been or arecurrently used for the prevention, treatment and/or management of aviral infection can be determined using references available to aclinician such as, e.g., the Physicians' Desk Reference (61^(st) ed.2007). Preferably, dosages lower than those which have been or arecurrently being used to prevent, treat and/or manage the infection areutilized in combination with one or more compounds or compositions.

For compounds which have been approved for uses other than prevention,treatment or management of viral infections, safe ranges of doses can bereadily determined using references available to clinicians, such ase.g., the Physician's Desk Reference (61^(st) ed. 2007).

The above-described administration schedules are provided forillustrative purposes only and should not be considered limiting. Aperson of ordinary skill in the art will readily understand that alldoses are within the scope of the invention.

It is to be understood and expected that variations in the principles ofinvention herein disclosed may be made by one skilled in the art and itis intended that such modifications are to be included within the scopeof the present invention.

Throughout this application, various publications are referenced. Thesepublications are hereby incorporated into this application by referencein their entireties to more fully describe the state of the art to whichthis invention pertains. The following examples further illustrate theinvention, but should not be construed to limit the scope of theinvention in any way.

EXAMPLES Example 1 Screen of Antiviral Targets

To identify host cell enzymes whose inhibition could decrease theproduction of virus progeny, an siRNA screen was performed to test forroles of specific enzymes involved directly or indirectly in lipidmetabolism. The siRNA screen was designed to test for effects ofinhibiting specific siRNA targets on the infectious yield of HCMV. Thescreen was performed in 96-well plates using a portion of an siRNAlibrary purchased from Sigma. Each target was assayed with threedifferent siRNAs. (See Table 2).

MRC5 human fibroblasts received siRNAs by transfection, 24 h later theywere infected at a multiplicity of 0.1 pfu/cell with an HCMV derivativeexpressing a GFP tagged protein, and 96 h after infection cells in eachwell were observed for spread of the GFP marker. Also at 96 h afterinfection, the medium was removed from each well and used to infectfibroblasts in a new 96-well plate, and infectivity was quantified bystaining for immunofluorescence and counting IE1-positive cells 24 hlater.

The siRNA which were found to inhibit HCMV replication are shown inTable 1. Note that, even when siRNA resulted in only a modest (e.g.˜2-fold) inhibition of viral replication, the associated enzyme may bepivotal for viral replication, as siRNA typically results in only amodest, e.g., 2- to 10-fold, reduction in enzyme activity. Furtherreduction in enzyme activity (e.g., using a more effective siRNA or asmall molecule inhibitor) may result in greater, sometimes much greater,inhibition of viral replication.

Example 2 Antiviral Effects and Theapeutic Index of Triacsin C

Triacsin C has been administered orally to mice for two months at 10mg/kg/day with no significant toxicities noted, and it significantlyinhibited the progression of atherosclerosis (LDLR^(−/−) mice) (Matsudaet al., J. Antibiot. 6:318-21, 2008). Also, WS-1228 A and B, compoundsstructurally related to triacsin C, exhibit vasodilator activity (Omuraet al., J Antibiot 39:1211-8, 1986).

The effect of triacsin C on the viability of MRC5 human fibroblasts inculture medium containing 10% fetal calf serum was tested. At the timedrug was added, cells were about 90% confluent. After 96 h the drug didnot alter the appearance of cells when administered at 100 or 250 nM,but caused some cell rounding at 500 nM (FIG. 2, top panels). The drughad little effect on cell viability at doses of ≦500 nM (FIG. 2, bottomleft). Triascin C was also tested for its effect on the viability ofMRC5 human fibroblasts in the absence of fetal calf serum, where itshowed no discernable impact on viability at doses up to 10 μM. Whenadded together with the infecting virus, triacsin C inhibited theproduction of HCMV in a dose-dependent manner, reducing the yield ofinfectious virus by a factor of 85 at 250 nM (FIG. 2, bottom right).

At 50 nM, triacsin C reduced the yield of infectious HCMV virus by afactor of >8-fold without discrenably impacting the viability of MRC-5fibroblasts in the presence of serum. Thus, triacsin C shows a favorabletherapeutic index as an antiviral of >10-fold. For fibroblasts in theabsence of serum, the therapeutic index is yet morefavorable, >100-fold. Based on the in vivo tolerability of 10 mg/kg/day,it is likely that the fibroblasts in the absence of serum are the morerelevant reflection of in vivo biology (the MRC-5 fibroblasts in theabsence of serum are quiescent, like most cells in vivo). Thus, triacsinC is anticipated to have an in vivo therapeutic index of >2-fold,preferably >5-fold or >10-fold, and more preferably >20-fold.

Lipid drop formation is associated with HCMV infection. Triacsin Ctreatment reduced lipid droplet formation induced by HCMV infection ofHFF cells (not shown).

Lipid droplet formation and depletion is associated with HCMV infection.At early phase of infection (2-12 hours after infection) lipid dropletsare induced within the infected cells and thereafter, a complete loss oflipid droplets is observed (48-72 hours after infection). On the otherhand, the cells that do not harbor HCMV but are neighboring to theinfected cells dramatically accumulate lipid droplets starting with 48hours after infection. Triacsin C treatment reduced lipid dropletformation induced by HCMV infection of HFF cells in both cases.

Triacsin C was tested for its ability to inhibit the growth ofadditional viruses. MRC5 fibroblasts were infected with HCMV, herpessimplex virus type 1 (HSV-1), adenovirus type 5 or influenza A andtreated with indicated amounts of triacsin C beginning when virus wasadded to cells (FIG. 3). All of the enveloped viruses (HCMV, HSV-1,influenza A) were substantially inhibited by drug treatment, whileadenovirus was not.

In addition, hepatitis C virus (HCV) growth was also inhibited byTriacsin C. Huh7.5 cells which are susceptible to HCV infection wasinfected with a derivative of JFH1 strain and treated with indicatedamounts of Triacsin C (FIG. 9). The release of infectious HCV particlesto the culture medium was significantly reduced by Triacsin C treatment.

Example 3 Antiviral Effects of Aminoxyacetic Acid (AOAA)

Replication of HCMV was tested in MRC5 fibroblasts. 0.5 mM AOAA resultedin a 100-fold decrease in viral replication (FIG. 4), with no measurabledecrease in cell viability at concentrations up to 2.5 mM (5-foldtherapeutic index for 100-fold antiviral effect).

Replication of influenza A (WSN/33 strain) was measured in MDCK cells ata multiplicity of infection of 0.001 particle forming unit per cell.There was no evidence of host cell toxicity at doses of 0.5 and 1 mMbased on light microscopy. Both of these doses markedly delayed thereplication of influenza A, decreasing viral yields at 24 hours postinfection by >1000-fold at each dose level (FIG. 5). Such a delay inviral spread is anticipated to enable effective immune clearance of thevirus and therefore to prevent, eliminate, or greatly mitigate diseasein an infected mammal.

Replication of adenovirus was tested in MRC5 fibroblasts at amultiplicity of infection of 1 particle forming unit per cell. 0.5 mMAOAA resulted in a 20-fold decrease in viral replication, and 1 mM AOAAresulted in a 500-fold decrease in viral replication (FIG. 6).

Example 4 Antiviral Effects of Meta-Iodo-Benzylguanidine (MIBG)

Replication of HCMV was tested in MRC5 fibroblasts. Cells were infectedwith HCMV and incubated at MIBG concentrations of 0 μM, 50 μM, 100 μM,and 250 μM. Virus yield in the medium at 96 hours after infection wasdetermined. 50 μM MIBG resulted in an approximately 70% decrease invirus titer, with little or no effect on cell morphology. (FIG. 7)

Example 5 Antiviral Effects of Simvastatin

Replication of HCMV was tested in MRC fibroblasts. Cells were infectedwith HCMV and incubated at simvastatin concentrations of 0 μM, 1 μM, 2.5μM, and 5 μM. Virus yield in the medium at 96 hours after infection wasdetermined. 5 μM simvastatin resulted in an approximately 90% decreasein virus titer. (FIG. 8)

Example 6 Antiviral Effects of PF-1052

Replication of HCMV (AD169 strain) was tested in MRC5 cells at amultiplicity of infection of 0.1 particle forming unit per cell. Therewas no evidence of host cell toxicity at doses of 5 and 10 μM based onlight microscopy. Both of these doses markedly reduced the expression oflate viral protein pUL99 coupled to GFP. PF-1052 reduced the viralyields at 96 hours post infection by >50-fold at 5 μM and by >300-foldat 10 μM (FIG. 10).

PF-1052 was also tested for its ability to inhibit HCMV in a prolongedtreatment of two weeks. The culture medium was replaced with freshmedium containing 5 μM PF-1052 or ethanol as a control every day. At 10days after infection, PF-1052 dramatically reduced the yield of HCMV bya factor of >5000 fold (FIG. 11). After 10 days, HCMV infection in thecontrol cells that was reduced due to the severe infection that causescell death while the virus yields remained constant in PF-1052 treatedcells. 14 days after infection, PF-1052 was removed and no longer addedto the cells. The virus production was substantially increased 2 and 5days after the removal of PF-1052, indicating that the inhibitory effectof the drug is specific and not due to a permanent change that occurs inthe cells during prolonged drug treatment.

PF-1052 was tested for its ability to inhibit the growth of additionalviruses. MRC5 fibroblasts were infected with HSV-1, adenovirus type 5 orinfluenza A and treated with indicated amounts of PF-1052 (FIG. 12-14).HSV-1 and influenza A which contain a viral envelope were substantiallyinhibited by drug treatment in a dose dependent manner, while adenoviruswas not.

In addition, HCV growth was also inhibited by PF-1052. Huh7.5 cellswhich was infected with HCV and treated with indicated amounts ofPF-1052 (FIG. 15). PF-1052 inhibited the release of infectious HCVparticles to the culture medium at 5 μM by a factor of ˜10 fold and at10 μM by a factor of >100 fold.

Example 7 Antiviral Effects of Rubimaillin

Replication of HCMV was tested in MRC fibroblasts. Cells were infectedwith HCMV and incubated with rubimaillin at a concentration of 10 μM.Virus yield in the medium at 96 hours after infection was determined. 5μM rubimaillin resulted in an approximately 85% decrease in virus titer.(FIG. 16)

Example 8 Antiviral Effects of Elongase Inhibitors

One class of elongase inhibitors is benzoxazinone compounds (Mizutani etal., J. Med. Chem., 52: 7289-7300, 2009). Among these the most potentcompound, (S)-1y shows specific inhibitory activity against human ELOVL6with an IC₅₀ of ˜2.6 nM and human ELOVL3 with an IC₅₀ of ˜130 nM invitro. Note that the plasma protein unbound fraction ratio of (S)-1y wasfound to be about 10% in tissue culture experiments which raises theexpected inhibitory concentration cells. In line with this, the in vivoIC50 values of (S)-1y was found to be 169 nM for ELOVL6 and >5 μM forELOVL3. Without inducing any toxicity on the cells, (S)-1y dramaticallyreduced HCMV late pUL99 expression in a dose dependent manner.Similarly, at 25 μM concentration, (S)-1y inhibited HCMV replication bya factor of ˜40 fold, and at 50 μM by a factor of >1000 fold (FIG. 17).Whereas, it's inactive isomers R-1y and racemic-1y did not show a potentinhibitory effect on virus replication.

Another class of elongase inhibitors is3-sulfonyl-8-azabicyclo[3.2.1]octane compounds (Nagase et al., J. Med.Chem., 52: 4111-4114, 2009). Among these, the endo isomer of compound 1kand 1w inhibit human ELOVL6 with an IC₅₀ of about 78 nM and 1w inhibitshuman ELOVL6 with an IC₅₀ of about 6.9 μM in vitro. The endoconfiguration of these particular compounds is essential for theirinhibitory action since exo isomers are devoid of potency. The compoundsEndo-1k and Exo-1w were tested for their effect on HCMV replication.MRC5 cells were infected with HCMV AD169 strain at a multiplicity of 0.1pfu per cell. Without inducing any toxicity on the cells, Endo-1kdramatically reduced HCMV late pUL99 expression and virus yield in adose dependent manner and at 50 μM it inhibited HCMV replication by afactor of ˜400 fold (FIG. 18). Expectedly, the compound Exo-1w neitheraffected late viral protein expression nor HCMV yield indicating thatantiviral function of Endo-1k is due to the inhibition of elongaseenzymes specifically.

Yet another class of elongase inhibitors is indoledione compounds whichare selective for ELOVL6 and ELOVL3 over other elongases (Takahashi etal., J. Med. Chem., 52: 3142-3145, 2009). Among these, Compound 37inhibits human ELOVL6 and ELOVL3 with IC₅₀ of ˜8.9 nM and ˜337 nMrespectively in vitro. The drug has been administrated orally to miceand at a dose of 10 mg/kg which resulted in 30 and 50 μM plasma andliver levels 2 hours after treatment. The effect of Compound 37 on HCMVreplication was tested in MRC5 cells. The cells were infected with HCMVAD169 strain at a multiplicity of 0.1 pfu per cell. Without inducing anytoxicity on the cells, at 35 μM concentration, compound 37 substantiallyreduced late viral pUL99-GFP expression which is determined byfluorescence microscopy (FIG. 19). Similarly, at 35 μM, compound 37inhibited HCMV replication by a factor of >10 fold, and at 70 μM, itreduced virus yield to undetectable levels.

The elongase inhibitor compounds indicated above are structurallydiverse and potent inhibitors of ELOVL6 and less potent inhibitorsELOVL3. The most potent inhibitor of each classes exerted similarantiviral effects on HCMV. Thus, the compounds that inhibits ELOVL6,ELOVL3 and both ELOVL6/ELOVL3 are identified as potent antivirals.

Example 9 Gene Expression Analysis of ACSLS in HCMV Infected Cells

HCMV infection causes global changes in cellular mRNA levels. Toevaluate whether any particular change in ACLS mRNA levels occurs inHCMV infection, the mRNA levels of all 5 ACSL isomers were analyzed byquantitative RT-PCR method. Among these 5 genes, mRNA levels of ACSL1were elevated at 48 h after infection (FIG. 20; upper panel). In linewith this, the analysis of ACSL1 protein levels showed a dramaticincrease of ACSL1 during the course of infection (FIG. 20; lowerpanels). This together with the fact that inhibition of ACSL1 by siRNAsor triacsin C blocks HCMV replication, points ACSL1 as a preferredtarget for treating HCMV infections. In addition, ACSL6 mRNA levels wereshown to be enhanced by HCMV infection even to a greater extent thanACSL1. Note that ACSL6 is not sensitive to triacsin C and these datapredict that siRNAs or small molecule inhibitors targeting ACSL6 arepotential antivirals.

Example 10 Enhanced Antiviral Effects of a Combination of an ACCInhibitor and an ELOVL Inhibitor

TOFA acts as an inhibitor of acetyl-CoA carboxylase (ACC), whichproduces malonyl-CoA, the substrate for both de novo fatty acidsynthesis by fatty acid synthase (FAS) as well as elongation ofacyl-CoAs by elongases of very long chain fatty acids (ELOVLs). The datapresented herein indicate that inhibition of ACC or specific ELOVLsalone suffice to interfere with the reproduction of a number ofenveloped viruses. The present example concerns the combined use of ACCinhibitors (e.g., TOFA) and ELOVL inhibitors to antagonize viralreplication and spread. A non-limiting example of a rationale for abeneficial antiviral effect of a combination of ACC inhibitor and ELOVLinhibitor follows from the fact that ACC catalyzes the rate-limitingreaction for both fatty acid synthesis and elongation, and ELOVLinhibitors directly interfere with the elongation process. Since ACCinhibition by TOFA directly inhibits malonyl-CoA production andindirectly inhibits palmitate production by depriving FAS of themalonyl-CoA needed for palmitate synthesis, it depletes both thesubstrates for the very long chain fatty acid elongation process, whichcanonically begins with palmitate. Together with a direct ELOVLinhibitor this would synergistically inhibit ELOVL activity, potentiallyallowing one or both drugs to be used at a lower dose and preventundesirable side effects. Non-limiting examples of assays that could beperformed to assess the effect of a combination of an ACC inhibitor andan ELOVL inhibitor on HCMV, influenza A, HBV, or HCV replication follow.Assays for TOFA-mediated antiviral activity using HCMV-infected humanfibroblasts, influenza A-infected MDCK cells, HIV-1-infected C8166cells, HBV-producing HepG2-2.2.15 cells, and Huh7 ET cells that containan HCV RNA replicon have been described in WO 2009/023059. In eachassay, various concentrations of TOFA can be combined with variousconcentrations of different ELOVL inhibitors and assayed for theireffect on virus replication. In one preferred embodiment, aphysiological concentration of an ELOVL inhibitor will be held constantas the dose of TOFA is increased. Control cultures are treated with nodrug, ELOVL inhibitor alone or the various concentrations of TOFA alone.Samples are taken at 24, 48, 72 and 96 hours after initiation of drugtreatment. The antiviral effect of ELOVL inhibitor plus eachconcentration of TOFA is then compared to the activity of ELOVLinhibitor alone or the various concentrations of TOFA alone. Uptake ofneutral red dye can be used to determine the relative level of toxicityin duplicate cultures at each time a sample is harvested. Theconcentration of TOFA required to reduce viral growth and/or replicationby 10-fold is reduced by 2-fold, and sometime more, by the presence of atherapeutically effective concentration of the ELOVL inhibitor.

Example 11 Antiviral Effects of TOFA

TOFA is an allosteric inhibitor of ACACA and has been shown to blockproduction of human cytomegalovirus progeny in cultured cells asdescribed in WO 2009/023059. TOFA was tested for its ability to inhibitHCMV replication. At a dose of 5 μg/ml, TOFA inhibited HCMV replicationby a factor of >500 fold.

In addition, HCV growth was also inhibited by TOFA. Huh7.5 cells whichwas infected with HCV and treated with indicated amounts of TOFA (FIG.21). TOFA inhibited the release of infectious HCV particles to theculture medium at 5 μg/ml by a factor of >500 fold and at 10 μg/ml by afactor of >1000 fold.

The following examples provide non-limiting examples of proposedcombinations of the present invention.

Example 12 Enhanced Antiviral Effects of a Combination of an ACCInhibitor and an ACSL1 Inhibitor

Acyl-CoA synthetase (long chain) member 1 (ACSL1) activates free fattyacids, such as the palmitate derived from de novo fatty acid synthesis,by conjugating them to coenzyme A. These acyl-CoAs are the substrate forthe majority of the lipid biosynthetic enzymes that act on acyl chains.The data presented herein indicate that inhibition of ACC or ACSL1 alonesuffices to interfere with the reproduction of a number of envelopedviruses. The present example concerns the combined use of ACC inhibitors(e.g., TOFA) and ACSL1 inhibitor (e.g., triacsin C) to antagonize viralreplication and spread. There are multiple rationales for such acombination. Non-limiting examples of such rationales include thefollowing. While viral infection can up-regulate the de novo fatty acidbiosynthetic, the major source for fatty acids in most mammalian celltypes consists of the circulating lipids present in the plasma.Therefore, a TOFA-induced blockade of fatty acid biosynthesis might bepartially or completely overcome through the influx of plasma fattyacids. Triacsin C co-treatment could prevent these exogenous fatty acidsfrom being activated and utilized, thereby enhancing the efficacy ofTOFA treatment. Conversely, triacsin C-induced ACSL1 inhibition might beovercome by the expression of a different, triacsin-resistant ACSL; forexample, ACSL5 overexpression was shown to rescue the inhibitory effectof triacsin C on glioma cells (Mashima et al., Cancer Sci. 2009100:1556-1562). This presents an infecting virus with a possibly hostcell-dependent means of escape from triacsin C inhibition. Simultaneoustreatment with TOFA, however, would deplete the substrates for all ACSLenzymes, and so prevent viral escape. Thus these drugs in combinationmight synergize so as to lower the effective dose of one or both, aswell as lower the occurrence of treatment failure.

Non-limiting examples of assays that could be performed to assess theeffect of a combination of an ACC inhibitor and an ACSL1 inhibitor onHCMV, influenza A, HBV, or HCV replication follow. Assays forTOFA-mediated antiviral activity using HCMV-infected human fibroblasts,influenza A-infected MDCK cells, HIV-1-infected C8166 cells,HBV-producing HepG2-2.2.15 cells, and Huh? ET cells that contain an HCVRNA replicon have been described in WO 2009/023059. In each assay,various concentrations of TOFA can be combined with variousconcentrations of triacsin C and assayed for their effect on virusreplication. In one preferred embodiment, a physiological concentrationof triacsin C is held constant as the dose of TOFA is increased. Controlcultures are treated with no drug, triacsin C alone or the variousconcentrations of TOFA alone. Samples are taken at 24, 48, 72 and 96hours after initiation of drug treatment. The antiviral effect oftriacsin C plus each concentration of TOFA is then compared to theactivity of triacsin C alone or the various concentrations of TOFAalone. Uptake of neutral red dye is used to determine the relative levelof toxicity in duplicate cultures at each time a sample is harvested. Inthe presence of a pharmacologically acceptable concentration of triacsinC, the concentration of TOFA required to produce a 10-fold reduction inHCMV replication is markedly reduced, from ^(˜)10 μg/mL to <5 μg/mL. At10 μg/mL of TOFA, the magnitude of the therapeutic effect is increasedfrom ^(˜)10-fold in the absence of triacsin C to ^(˜)100-fold in itspresence. The combined use of TOFA and triacsin C does not increase hostcell toxicity as measured by the neutral red dye assay. Similar resultsare obtained for other triacsins and structurally related ACSL1inhibitors.

Example 13 Enhanced Antiviral Effects of a Combination of an ACCInhibitor and an Inhibitor of Viral Neuraminidase

Neuraminidase inhibitors are a critical component of the currentstandard of care for acute infection by influenza A. Thesevirally-encoded enzymes cleave sialic acid residues from glycoproteinson the host cell surface, freeing the recently replicated virion todiffuse away and invade another cell. Neuraminidase inhibitors such asoseltamivir (TAMIFLU™) block this process, thereby blocking viralrelease. The present example concerns the combined use of ACC inhibitors(e.g., TOFA) and neuraminidase inhibitors (e.g., oseltamivir, zanamivir)to antagonize viral replication and spread. A non-limiting example of arationale for a beneficial antiviral effect of a combination of ACCinhibitor and neuraminidase inhibitor follows from the fact that thesedrugs target different but equally necessary processes in viralreplication: virion assembly and virion release. They would therefore beexpected to act synergistically to inhibit viral spread, e.g., if TOFAtreatment alone results in a ten-fold reduction in viable virionsassembled, and oseltamivir treatment alone causes a ten-fold reductionin the virion release, then the combination would result in ahundred-fold reduction in the release of viable virions.

Non-limiting examples of assays that could be performed to assess theeffect of a combination of an ACC inhibitor and a neuraminidaseinhibitor on influenza A replication follow. Assays for TOFA-mediatedantiviral activity using influenza A-infected MDCK cells have beendescribed in WO 2009/023059. In each assay, various concentrations ofTOFA can be combined with various concentrations of oseltamivir andassayed for their effect on virus replication. In one preferredembodiment, a physiological concentration of oseltamivir is heldconstant as the dose of TOFA is increased. Control cultures are treatedwith no drug, oseltamivir alone or the various concentrations of TOFAalone. Samples are taken at 24, 48, 72 and 96 hours after initiation ofdrug treatment. The antiviral effect of oseltamivir plus eachconcentration of TOFA is then compared to the activity of oseltamiviralone or the various concentrations of TOFA alone. Uptake of neutral reddye is used to determine the relative level of toxicity in duplicatecultures at each time a sample is harvested. In the presence of apharmacologically acceptable concentration of oseltamivir, theconcentration of TOFA required to produce a 10-fold reduction in HCMVreplication is markedly reduced, from ^(˜)10 μg/mL to <5 μg/mL. At 10μg/mL of TOFA, the magnitude of the therapeutic effect is increased from^(˜)10-fold in the absence of oseltamivir to ^(˜)100-fold in itspresence. The combined use of TOFA and oseltamivir does not increasehost cell toxicity as measured by the neutral red dye assay. Similarresults are obtained for other neuraminidase inhibitors.

Example 14 Enhanced Antiviral Effects of a Combination of an ACCInhibitor and a Viral Entry Inhibitor

One key step in the HIV-1 life cycle consists of the virion'srecognizing, binding to, and finally fusing with the correct host cell.This is the process by which newly-synthesized virions are able toinvade a new host cell, thereby spreading the infection. Viral entry isso central to the pathology of HIV-1 infection that pharmacologicalinhibition of binding and fusion events has proven a viable strategy inthe clinical management of the disease. The present example concerns thecombined use of ACC inhibitors (e.g., TOFA) and entry inhibitors (e.g.,maraviroc, enfurtivide) to antagonize viral replication and spread. Anon-limiting example of a rationale for a beneficial antiviral effect ofa combination of ACC inhibitor and neuraminidase inhibitor follows fromthe fact that these drugs target different but equally necessaryprocesses in viral replication: virion assembly and virion entry. Theywould therefore be expected to act synergistically to inhibit viralspread, e.g., if TOFA treatment alone results in a ten-fold reduction inviable virions assembled, and enfurtivide treatment alone causes aten-fold reduction in the successful entry of viable virions, then thecombination would result in a hundred-fold reduction in the entry ofviable virions.

Non-limiting examples of assays that could be performed to assess theeffect of a combination of an ACC inhibitor and an entry inhibitor onHIV-1 replication follow. Assays for TOFA-mediated antiviral activityusing HIV-1-infected C8166 cells have been described in (WO2009/023059). In each assay, various concentrations of TOFA can becombined with various concentrations of enfurtivide and assayed fortheir effect on virus replication. In one preferred embodiment, aphysiological concentration of enfurtivide is held constant as the doseof TOFA is increased. Control cultures are treated with no drug,enfurtivide alone or the various concentrations of TOFA alone. Samplesare taken at 24, 48, 72 and 96 hours after initiation of drug treatment.The antiviral effect of enfurtivide plus each concentration of TOFA isthen compared to the activity of enfurtivide alone or the variousconcentrations of TOFA alone. Uptake of neutral red dye is used todetermine the relative level of toxicity in duplicate cultures at eachtime a sample is harvested. In the presence of a pharmacologicallyacceptable concentration of enfurtivide, the concentration of TOFArequired to produce a 10-fold reduction in HCMV replication is markedlyreduced, from ^(˜)10 μg/mL to <5 μg/mL. At 10 μg/mL of TOFA, themagnitude of the therapeutic effect is increased from ^(˜)10-fold in theabsence of enfurtivide to ^(˜)100-fold in its presence. The combined useof TOFA and enfurtivide does not increase host cell toxicity as measuredby the neutral red dye assay. Similar results are obtained for otherentry inhibitors.

We claim: 1-103. (canceled)
 104. A pharmaceutical composition fortreatment or prevention of a viral infection comprising atherapeutically effective amount of a compound or prodrug thereof, orpharmaceutically acceptable salt of said compound or prodrug; and apharmaceutically acceptable carrier, wherein the compound is a compoundof i) Formula VI:

wherein L is selected from carbamate, urea, or amide including, forexample

and wherein R is selected from halo; CF₃; cyclopropyl; optionallysubstituted C₁₋₅ alkyl, wherein the C₁₋₅ alkyl is substituted with halo,oxo, —OH, —CN, —NH₂, CO₂H, and C₁₋₃ alkoxy; wherein R₁ is selected fromsubstituted phenyl where the substituents are selected from F, CF₃, Me,OMe, or isopropyl; wherein R₂ is Cl, Ph, 1-(2-pyridone), 4-isoxazol,3-pyrazol, 4-pyrazol, 1-pyrazol, 5-(1,2,4-triazol), 1-(1,2,4-triazol),2-imidazolo, 1-(2-pyrrolidone), 3-(1,3-oxazolidin-2-one); and whereinthe chiral center at C4 is racemic, (S), (R), or any ratio ofenantiomers; ii) Formula VIIa or VIIb:

wherein R₁ is selected from OMe, OiPr, OCF₃, OPh, CH₂Ph, F, CH₃, CF₃,and benzyl; and wherein R₂ is selected from C₁₋₄ alkyl; phenyl;substituted phenyl where substitutents are selected from OMe, CF₃, F,tBu, iPr and thio; 2-pyridine; 3-pyridine; and N-methy imidazole; iii)Formula VIII

wherein R₁ is selected from H, unsubtitued phenyl; substituted phenylwhere substitutents are selected from F, Me, Et, Cl, OMe, OCF₃, and CF₃;C₁₋₆ alkyl; and C₃₋₆ cycloalkyl; wherein R₃ and R₄ are independentlyselected from H; C₁₋₃ alkyl; and phenyl; or R₃ and R₄ taken togetherform a cycloalkyl of formula —(CH2)_(n)— where n=2, 3, 4 and 5; whereinR₅ is selected from methyl; CF₃; cyclopropyl; unsubtitued phenyl; mono-and disubstituted phenyl where substitutents are selected from F, Me,Et, CN, iPr, Cl, OMe, OPh, OCF₃, and CF₃; unsubstituted heteroaromaticgroups; and imidazole; and iv) Formula IX:

wherein L is selected from urea, amide,

wherein R₁ is selected form 2-, 3-, and 4-pyridine; pyrimidine;unsubstituted heteroaryls such as isoxazol, pyrazol, triazol, imidazole;and unsubstituted phenyl; ortho, meta or para-substituted phenyl wheresubstitutents are F, Me, Et, Cl, OMe, OCF₃, and CF₃, Cl, iPr and phenyl;and wherein R₂ is selected from Cl; iPr; phenyl; ortho, meta orpara-substituted phenyl where substitutents are F, Me, Et, Cl, OMe,OCF₃, and CF₃; and heteroaryls such as 2-, 3-, and 4-pyridine,pyrimidine, and isoxazol, pyrazol, triazol, and imidazo.
 105. Thepharmaceutical composition of claim 104, wherein the compound of FormulaVI is


106. The pharmaceutical composition of claim 104, wherein the compoundof Formula VIIa and VIIb is


107. The pharmaceutical composition of claim 104, wherein R⁵ in thecompound of Formula VIII is


108. The pharmaceutical composition of claim 104, wherein the compoundof Formula VIII is


109. The pharmaceutical composition of claim 104, wherein the compoundof Formula IX is